WO2011021779A2

WO2011021779A2 - Method for evaluating water quality and toxicity using the growth rate of germ tubes of spores of algae belonging to the order laminariales

Info

Publication number: WO2011021779A2
Application number: PCT/KR2010/004682
Authority: WO
Inventors: 한태준; 공정애
Original assignee: 인천대학교 산학협력단
Priority date: 2009-08-19
Filing date: 2010-07-19
Publication date: 2011-02-24
Also published as: WO2011021779A3; KR101084761B1; KR20110019066A

Abstract

The present invention relates to a method for evaluating water quality and toxicity using the reactivity of spores of algae belonging to the order Laminariales against toxic materials. The method of the present invention for evaluating water quality and toxicity comprises the steps of: putting a water sample into a measuring container; adding spores of algae belonging to the order Laminariales to the water sample in the measuring container; culturing the spores added into the measuring container; and measuring the germination rate of the cultured spores or measuring the growth rate of germ tubes of spores germinated from the cultured spores. The method for evaluating water quality and toxicity using the reactivity of spores of algae belonging to the order Laminariales against toxic materials according to the present invention can enable the quick evaluation of water quality and toxicity within 24 hours, as well as the effective measurement of toxic materials in a water sample, including chloride or a high salt content. Especially in the case of the method using the growth rate of germ tubes of spores as a standard for evaluating water quality and toxicity, the sensitivity against toxic materials can be enhanced as compared to the method for using the germination rate of spores. In addition, since a method for prolonged preservation of green laver has not yet been developed, the method of the present invention using Laminariales, which can preserve biological samples for more than 14 months, can be very progressive not only as a diagnostic technique for ecological toxicity but also as a process for establishing a kit.

Description

Evaluation of Water Toxicity Using Germination Tube Growth Rate of Seaweed Spores in Sea Tangle

The present invention relates to a method for evaluating water toxicity using the reactivity of toxic substances of seaweed spores belonging to kelp, and more particularly, to a method for evaluating water toxicity using germination rate or germination tube growth rate of seaweed spores belonging to kelp. will be. In addition, the present invention relates to a water toxicity assessment kit comprising algae spores belonging to the seaweed

Recently, as a method for improving the water pollution problem, which is mentioned as one of the environmental problems, there is increasing interest in the method of improving the water quality and monitoring the leakage of harmful substances in rivers and the like.

Conventional methods for detecting toxic substances that may enter the water system include physicochemical and biological methods. Physicochemical methods include gas chromatography (GC) and inductively coupled plasma. However, these methods require expensive analytical equipment such as gas chromatography-mass spectroscopy (GC-MS) and inductively coupled plasma-mass spectroscopy (ICP-MS), and highly skilled analytical personnel. There is a problem that must be performed. In addition, when the analysis time is long, and the components of the influx of toxic substances are unknown, there is a problem that is difficult to analyze, and when the sample is moved over a long distance, there is a problem that the components and concentration change may be caused by the reaction in the sample.

Due to these shortcomings, biological methods that use organisms as biomarkers for toxic substances have emerged, and biological methods include microbial metabolism or enzymes such as bacteria, or shellfish, invertebrates, microalgae or protozoa. Etc. There is a method using eukaryotes. Toxicity investigation using microorganisms is a method of measuring growth inhibition, oxygen consumption rate, colon formation rate, ATP concentration, enzyme activity, lethality (or survival rate) or bioluminescence.

In general, in the case of using fish or invertebrates, it is judged by mortality rate, movement inhibition, and biological characterization. In case of using microalgae, growth inhibition is measured. Daphnia magma and Ceriodaphnia dubia , Daphnia , are very sensitive to environmentally toxic substances and are easy to cultivate, have short lifespan and small biomass, and can be used for experiments. It is used. However, the method of using such fish, invertebrates or protozoa requires the expert's expertise and expensive equipment, and the sensitivity varies depending on the toxic substances. Especially, if the base technology for cultivating the experimental organism is not equipped, There is a problem that the countermeasures follow. In addition, daphnia are very vulnerable to chlorine, and all of them are killed in the effluent containing trace amounts of chlorine, which causes a serious problem that cannot be determined.

On the other hand, Korean Patent Nos. 10-0653100 and 10-0653101 disclose a method for evaluating water toxicity by using the spore formation rate of the seaweed, in order to determine the spore formation rate of significant seaweed culture for at least 96 hours There is a limit to the rapid determination of water toxicity.

The present invention is to solve the conventional problems, one object of the present invention is to have a stable reaction sensitivity to a water sample containing chlorine, even in a water sample having a high salt concentration is sensitive to a toxic substance, and discriminating It is to provide a method for evaluating water toxicity in a very short time.

Another object of the present invention is to provide a method for evaluating water toxicity with improved reaction sensitivity to metal toxic substances or volatile organic compound toxic substances.

Still another object of the present invention is to provide a water toxicity test kit that can implement the water toxicity test method according to the present invention.

In order to solve the object of the present invention, the present invention comprises the steps of putting a water sample in a measuring container; Injecting spores of algae belonging to the kelp in a measuring container containing a water sample; Culturing the spores put into the measuring container; And measuring the germination rate of the cultured spores. In addition, in order to solve the other object of the present invention, the present invention comprises the steps of putting a water sample in a measuring vessel; Injecting spores of algae belonging to the kelp in a measuring container containing a water sample; Culturing the spores put into the measuring container; And measuring the germination tube growth rate of the spores germinated in the cultured spores.

The germination rate of cultured spores is calculated as the ratio of the number of spores germinated to the number of cultured spores, and the germination tube growth rate of germinated spores is calculated as the germination tube average length of germinated spores. Germinated spores or germinated tube average lengths of germinated spores were measured using a microscope equipped with a condenser for condensing light. Specifically, by converting an image of a spore from a color close to white to a color close to black), the number of germinated spores or the average length of germination tubes of the germinated spores can be easily measured. In addition, spores may be cultured regardless of the light irradiation amount, it is characterized in that cultured at a temperature of 10 ~ 20 ℃ in the dark state.

In order to achieve another object of the present invention, the present invention is spores of seaweed belonging to the kelp; A measuring container for injecting the water sample and the spores of the seaweed and culturing the spores of the seaweed; And artificial saline with a salt concentration of 25-45 ‰ to dilute the water sample; It provides a water toxicity assessment kit comprising a. The water toxicity assessment kit according to the present invention may further include a standard toxic substance solution prepared by dissolving a single toxic substance in an artificial saline having a salt concentration of 25 to 45 ‰. It can be used to test the state of the spores, ie, the sensitivity of the reaction to toxic substances before evaluating them.

The water toxicity evaluation method using the reactivity of the toxic substances of seaweed spores belonging to the kelp according to the present invention, since the spore germination takes place in 24 hours, water toxicity compared to the water toxicity evaluation method using the conventional green spore formation rate Can be evaluated quickly. In addition, since the spores of seaweeds belonging to the sea tangle are more stable and reactive to chlorine, which is toxic than water fleas, it is reliable and accurate when using the germination rate or germination tube growth rate of seaweed spores belonging to the sea tangle as an assessment of water quality. Water toxicity can be assessed. In particular, when the spore germination tube growth rate is used as the water quality toxicity evaluation criterion, response sensitivity to toxic substances may be improved than when the spore germination rate is used. From the standpoint that long-term storage and mass culture of test organisms and homeostasis of provision determine the level of ecotoxicology techniques, kelp techniques that can store test organisms for more than 14 months are now compared to the seaweed technique, which has not yet been developed for long-term storage methods. This is not only an ecotoxicological diagnostic technique but also a very advanced technology in the kitting process.

FIG. 1 shows the images of the sea tangle spores before incubation and the spores incubated for 24 hours in seawater containing no toxic substances before and after adjusting the condenser of the microscope, and FIG. 2 shows copper (Cu) as a single toxic substance. After culturing the spores of kelp for 24 hours in a toxic solution containing (the initial copper concentration is 0.4 mg / L) and the toxic solution diluted by half dilution (cultivation conditions are 35 ‰ culture solution, 15 ℃, cancer) The image observed under the microscope is shown.

3 is a view schematically showing a water toxicity evaluation method according to an embodiment of the present invention.

Figure 4 shows the germination rate of kelp spores under various culture conditions, Figure 5 shows the germination tube average length of the kelp spores under various culture conditions.

Figure 6 is a graph showing the germination rate of kelp spores according to the concentration of toxic substances of metals, Figure 7 is a graph showing the germination tube average length of the kelp spores according to the concentration of toxic substances of metals.

8 is a graph showing the germination rate of kelp spores according to the concentration of toxic substances of volatile organic compounds, Figure 9 is a graph showing the germination tube average length of the kelp spores according to the concentration of toxic substances of volatile organic compounds.

An aspect of the present invention relates to a method for evaluating water toxicity, which has a stable reaction sensitivity to a chlorine-containing water sample, has a high sensitivity to a toxic substance even in a water sample having a high salinity concentration, and has a very short discrimination time. Water toxicity evaluation method according to an aspect of the present invention comprises the steps of putting a water sample in a measuring container; Injecting spores of algae belonging to the kelp in a measuring container containing a water sample; Culturing the spores put into the measuring container; And measuring the germination rate of the cultured spores. In addition, another aspect of the present invention relates to a method for evaluating water toxicity with improved reaction sensitivity to metal toxic substances or volatile organic compound toxic substances, and the method for evaluating water toxicity according to another aspect of the present invention includes a water sample in a measuring container. Putting a step; Injecting spores of algae belonging to the kelp in a measuring container containing a water sample; Culturing the spores put into the measuring container; And measuring the germination tube growth rate of the spores germinated in the cultured spores. Hereinafter, the water toxicity evaluation method according to the present invention will be described by dividing by component.

Water 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 water sample contains. The water sample is adjusted to have a salt concentration in the range of 15 to 55 ‰, preferably 25 to 45 ‰, for the satisfactory cultivation of seaweed spores belonging to the kelp prior to being placed in the measuring vessel, usually from seawater. Since the collected water sample satisfies the salt concentration described above, no adjustment is necessary, but in the case of fresh water, the salt concentration needs to be adjusted.

The first water sample (hereafter referred to as raw water) was adjusted to have a salt concentration in the range of 15 to 55 ‰, preferably 25 to 45 ‰, and then to a salt concentration in the range of 15 to 55 ‰, preferably 25 to 45 ‰. It is preferably diluted with artificial brine, 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)]. Artificial brine for diluting raw water is not particularly limited as long as it has a salt concentration in the range of 15 to 55 ‰, preferably 25 to 45 ‰, and if the salt concentration is the same as the adjusted salt concentration of the raw water, Can be. The raw water and diluted aqueous sample have a pH of 5-9, preferably 6-9, for smooth cultivation of algae spores belonging to kelp prior to being placed in a measuring vessel.

In the present invention, in the case of culturing seaweed spores belonging to kelp, using four or more water samples diluted in addition to raw water whose salt concentration is adjusted, the germination rate or growth rate of spores according to the diluting concentration of raw water can be obtained. Based on this, a single toxicity is more effective than half of the effective concentration (spore germination rate or growth rate of spores in the control group containing no toxic substance 50% less than the spore germination rate or growth rate in the control group containing no toxic substance). Specified at specific concentrations for substances and dilution rates for unknown raw water; Half maximal effective concentration (EC ₅₀ ) to no effect concentration (sprout germination rate to growth rate of spores in controls that do not contain toxic substances). Concentrations effective to maintain levels that are not significantly different from the growth rate For a single toxic substance, it is expressed at a certain concentration and for unknown raw water as a dilution rate; No Observed Effect Concentration (NOEC) can be evaluated.

Measuring vessel

The measuring container includes a water sample and spores of algae, and cultivates spores of algae, and the form thereof is not particularly limited, and an example is a well plate. The well plate is preferably composed of at least six wells. In one well, sprinkle spores with artificial saline containing no toxic substance as a control, and the remaining five wells are diluted with raw water and raw water. Add four water samples and incubate the spores.

Spores of algae belonging to kelp

The present invention uses spores of algae belonging to the kelp as a biomarker for the evaluation of water toxicity, and more specifically, the germination rate of the spores to the growth rate of germinated spores is used as an evaluation criteria of water toxicity. Since the spore germination takes place within 24 hours, the water toxicity evaluation method using the reactivity of the toxic substances of seaweed spores belonging to the kelp is known. Water toxicities can be assessed quickly, compared to 96 hours). In addition, the Daphnia method, which uses daphnia as a biomarker, has the disadvantage of not being able to evaluate chlorine toxicity because daphnia dies in water samples containing trace amounts of chlorine. Since the stability and reaction sensitivity to the substance chlorine is excellent, the water toxicity evaluation method according to the present invention can accurately evaluate chlorine toxicity. In particular, when using the germination tube growth rate of the spores germinated by the water toxicity evaluation criteria, the response sensitivity to toxic substances can be significantly improved than when using the germination rate of the spores.

Hereinafter, the characteristics of the present invention will be described more clearly by comparing the life history of seaweeds belonging to the kelp family according to the present invention and the life history of green seaweed.

(1) Life history of green seaweed and method for evaluating water toxicity using the same

Life cycle of the green sea "① spore formation (asexual reproduction) and spore release at the edge of the spores (green) ⇒ ② spores are released as spores and spouse formation (oil reproduction) ⇒ ③ male and female spouses are joined Formation and splicer growth to form a spore body (green sea) ", such as, the existing water quality toxicity evaluation method using the spore formation rate of the seaweed using step ① above. More specifically, in a green, uncontaminated environment, ① stage of reproduction (formation and release of spores) proceeds, and the color change of the leaf body called leaf appears. At first, the light greenish leaf body begins to reproduce (asexual reproduction). It turns dark olive and finally the spores turn white. However, in contaminated or toxic waters, fertility decreases and the degree of color change is reduced. Based on this principle, after exposing the green leaf to the toxic source, the leaf (leaf) is visually observed or the image is captured by a camera, and a scale magnifier is used to measure the area of the leaf showing the color change to white. In addition, the degree of toxicity of the toxin is indicated by comparison with the area of white leaf in the control group that does not contain the toxin. At this time, at least 96 hours are required for the formation and release of the spores of the green.

(2) Life history of algae belonging to kelp

Life history of algae belonging to the kelp family "releases spores from the sleeping bag made in the spore body (Tashima) ⇒ ② The released spores grow into spores (growth after germination) ⇒ ③ The female spores develop into eggs, the spores form eggs and the spores form spouses, and the spouses released are spliced with the eggs formed on the female spores. Formation of the conjugate and the development of the spores (Damashima) to go through the same step, "water toxicity evaluation method according to the present invention by using the step ② above, so that the spores in kelp life history can only be observed under a microscope The germination and growth timing of the spores, which are small-sized periods, are used as a measurement parameter or an end point.The germination and germination of the algae spores belonging to the kelp family takes place in about 24 hours. When using the water toxicity assessment method, the water toxicity assessment method using the spore formation and release of the green sea Water toxicity can be assessed quickly compared to cows.

Algae that can be used in the water toxicity assessment method according to the invention so long as it has the same life history and life of algae belonging to dasimagwa not greatly limited, specifically, Ecklonia cava (Ecklonia cava), more seaweed (Kjellmaniella crassifolia), gompi (Ecklonia stolonifera ), Seaweed ( Undaria pinnatifida ), or Chamdashima ( Laminaria. Japonica ).

Culture of Spores

Seaweeds belonging to the kelp family, more specifically, spores of Laminaria (jaminica) are characterized in that they are cultured in a cancerous state. In general, algae including the seaweed requires an appropriate dose of light to be cultured. Since the algae spores belonging to the kelp family according to the present invention are smoothly grown in the germination and germination tubes regardless of the amount of light, There is no need to worry about the amount of light.

The culture temperature for germination and germination tube growth of seaweed spores belonging to the kelp family is in the range of 5 ~ 25 ℃, preferably 10 ~ 20 ℃. In particular, the culture temperature for germination tube growth is more preferably in the range of 15 ~ 20 ℃.

The pH of the culture medium for germination and germination tube growth of seaweed spores belonging to the kelp family is in the range of 5-9, preferably 6-9. In addition, the salinity of the culture medium is in the range of 15 to 55 ‰, preferably 25 to 45 ‰. In particular, the pH of the culture medium for germination tube growth is more preferably in the range of 35 ~ 45 ‰.

Measurement of Spore Germination and Growth Rate of Germinated Spores

Spores released from adult seaweeds belonging to the kelp family lose their motility and adhere to the substrate, and then a part of the spores forms a tube, which is called a germ tube. The germination tube grows in length, along which the cytoplasm moves to the end of the germination tube. At this time, when the tube is formed from the spores is defined as "germination" (Germination), and if the tube is not formed it is determined that the germination.

After about 24 hours incubation of spores into seaweeds belonging to the kelp family, germination of spores and growth of germination tube are achieved, and the germination rate of cultured spores is calculated as the ratio of the number of germinated spores to the number of cultured spores. The germination tube growth rate of is calculated as the germination tube average length of germinated spores, and the calculated value can be compared with the germination rate or germination tube average length of spores cultured in a control group containing no toxic substances to evaluate the toxicity of the water sample. Can be. At this time, the number of germinated spores or germinated tube average length of the germinated spores is measured using an image analysis device. Preferably, the image analysis device is a microscope equipped with a condenser for condensing light. When the brightness of the cultured spore image is reduced by adjusting the microscope condenser (specifically, the image of the cultured spores is white). By changing from a color close to black to a color close to black), one can easily determine the number of spores germinated or the average length of germination tubes of germinated spores.

Figure 1 shows the image of the seaweed spores before incubation and the spores incubated for 24 hours in seawater containing no toxic substances before and after adjusting the condenser of the microscope. As shown in FIG. 1, the kelp spores show rapid change in germination within 24 hours in a non-contaminated environment, and specifically, a germ tube having a thread shape of about 25 μm in a circular spore is formed. . However, in an environment contaminated with toxic substances, germination ability is reduced and germination tube growth rate (germination tube length) is also reduced. On the other hand, when the spores of kelp germinate, they form a germination tube close to white, and when the condenser of the microscope is not controlled, it is displayed as an image close to white, which may cause difficulty in observing the germination and germination tube length of the spores. In this case, by adjusting the condenser of the microscope to convert the image of the cultured spores from a color close to white to a color close to black can easily observe the germination and germination tube length of the spores.

Assessment of Water Toxicity

Water quality toxicity of the water sample was evaluated by comparing the germination rate of spores cultured in raw water and diluted water sample or the germination rate of germinated tube growth of germinated spores with the germination rate or average length of germination tube grown in the control without toxic substances. Specifically, it is expressed as a value of Half maximal effective concentration (EC ₅₀ ) to No Observed Effect Concentration (NOEC). In this case, when the spore germination tube growth rate is used as the water quality toxicity evaluation criterion, the response sensitivity to the toxic substance is improved than when the spore germination rate is used.

Half maximal effective concentration (EC ₅₀ ) refers to the concentration of a water sample that is effective to reduce the germination rate or growth rate of spores to 50% less than the germination rate or growth rate of spores in a control free of toxic substances. For a water sample containing a single toxic substance, it is expressed as a specific concentration of a single toxic substance, and for an unknown source containing multiple toxic substances, it is expressed as the dilution rate of the raw water. No effect concentration means the concentration of the water sample effective to maintain the germination rate and growth rate of the spores at a level not significantly different from the germination rate or growth rate of the spores in the control group containing no toxic substances. Likewise, a water sample containing a single toxic substance is expressed at a certain concentration and for unknown raw water containing a large number of toxic substances, it is expressed as a dilution rate. On the other hand, when the results of the water toxicity evaluation method is expressed as half the effective concentration, the problem occurs that the toxicity of the actual water sample decreases as the size and the half effective concentration become larger. It can be expressed as:

TU = 100 / half effective concentration (%)

FIG. 2 shows a toxic solution containing copper (Cu) as a single toxic substance (initial copper concentration is 0.4 mg / L) and cultivated spores of kelp in a toxic solution diluted by half dilution for 24 hours (cultivation conditions 35 ‰ culture solution, 15 ° C., cancer state). Referring to Figure 2 describes the method for evaluating the effective half-effective concentration of the water sample containing a single toxic substance as follows. If the germination rate of spores is the basis for the evaluation of water toxicity, the number of spores cultured and the number of germinated spores in the control (copper concentration of 0 mg / L), the initial toxic solution, and the diluted 4 toxic solutions were measured, respectively. The percentage of total spores divided by the number of spores germinated is taken as the germination rate in each toxic solution. In general, the germination rate of the control group is 100%. Afterwards, the germination rate of each toxic solution and the control rate of germination in the control group were treated statistically to regard the copper concentration of the toxic solution having a germination rate of 50% compared to that of the control group as a half effective value, and statistically significant with the germination rate of the control group. The maximum copper concentration value of the toxic solution with no difference in germination rate is considered as no effect concentration. On the other hand, when the growth rate of the germinated spores is a criterion for evaluating the water toxicity, the same number of germinated spores extracted from the control group and the spores cultured in each toxic solution were extracted into the population, and instead of the number of germinated spores, The average length of germination spores of germinated spores was measured, and the average length of germination tubes in the toxic solution and the average length in the control group were statistically calculated to determine the copper concentration value of the toxic solution having an average length of 50% of the average length of the control group. And the maximum copper concentration value of the toxic solution having an average length that is not statistically significant different from the average length of the control group is considered as no effect 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 spore of kelp, and an incubator (a container for measuring). It can also be implemented by a ubiquitous system (remote control system) that takes pictures, sends them, analyzes them, and returns the results back. Toxic substances that can be evaluated by the water toxicity evaluation method of the present invention include cadmium (Cd; Cadmium), cobalt (Co; Cobalt), chromium (Cr; Chromium), copper (Cu; Copper), mercury (Hg; Mercury), Toxic substances in metals such as nickel (Ni; Nickel), lead (Pb), and zinc (Zn; Zinc), acetone, chloroform, dimethyl sulfoxide (DMSO), and ethyl alcohol ( It includes toxic substances of volatile organic compounds (VOCs) such as ethyl alcohol, formalaldehyde, methyl alcohol and phenol.

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.

In addition, since kelp is widely distributed all over the world and is similar in germination process, it is easy to systemize it using domestic kelp, and it contributes to the development of aquaculture industry by using kelp, a major aquaculture species of Korea, as a plant for evaluating water toxicity. can do.

Another aspect of the present invention relates to a water toxicity test kit capable of implementing the water toxicity test according to the present invention, wherein the water toxicity test kit according to the present invention comprises spores of seaweeds belonging to kelp; A measuring container for injecting the water sample and the spores of the seaweed and culturing the spores of the seaweed; And artificial saline with a salt concentration of 25-45 ‰ to dilute the water sample; It includes. In addition, the water toxicity test kit according to the present invention is cadmium (Cd; Cadmium), cobalt (Co; Cobalt), chromium (Cr; Chromium), copper (Cu; Copper), mercury (Hg; Mercury), nickel (Ni; Nickel, Pb (Lead), Zinc (Zn; Zinc), Acetone (acetone), Chloroform, Dimethyl sulfoxide, Ethyl alcohol, Formalin, Methyl A standard toxic substance solution prepared by dissolving any toxic substance selected from the group consisting of alcohols and phenols in artificial saline having a salt concentration of 25 to 45 ‰ may be further included. Toxic solutions can be used to test the state of spores, ie, the sensitivity of reactions to toxic substances, before assessing the water toxicity of a water sample. Specifically, when the standard toxic substance solution according to the present invention is a solution containing copper as a toxic substance, the data on the germination rate of the spores from the various copper solutions and the control group to the average length of the germination tube is obtained in advance, along with the water toxicity assessment kit. Can provide. The user measures the germination rate of the spores to the copper solution to the average length of the germination tube and compares it with the data provided in advance to determine whether the spores normally react to the toxic substances and determine a range (e.g., intrinsic response sensitivity). If more than 80% of the response sensitivity is known, you can proceed to assess water toxicity for unknown water samples.

After culturing spores of algae belonging to the kelp using the water quality toxicity assessment kit according to the present invention, whether the spores germinate and average germination tube length can be measured through a microscope equipped with a condenser for condensing light Can be. In addition, a microscope equipped with a condenser for condensing the light may be included in the water toxicity evaluation kit according to the present invention.

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

1. 다시마 포자의 발아 및 발아관 생장 유도를 위한 최적 배양 조건 확립1. Establishment of Optimal Culture Conditions for Germination of Germ Spores and Induction of Germination Tube Growth

(1) preparation of kelp spores

After cutting the spores from the leaves of Laminaria japonica , the complexes attached to the surface were wiped off with a kitchen towel, and the surfaces were washed again by putting the leaves in sea water. After the surface of the leaf was wiped dry, stored for 12 hours in the dark, the leaf was taken out and placed in a beaker containing seawater to induce the release of motility spores. The induced spore solution was placed in a Petri dish containing 20 ml of OTT's artificial seawater. At this time, a cover glass was placed on the bottom of the petri dish to serve as an attachment substrate, and a cover glass with spores was prepared by maintaining the culture in a constant temperature incubator (10 ± 0.5 ° C.) for 1 hour.

Table 1 shows the components and concentrations of the salts contained in the artificial seawater OTT's artificial seawater used in the embodiment of the present invention, OTT's artificial seawater has a salt concentration of 35 ‰.

Table 1

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

(2) cultivation of kelp spores

After spore-covered glass was put in a 24-well plate, the lid was closed and transferred to the incubator, and incubated for 24 hours under various light irradiation doses, pH of the culture solution, salt concentration of the culture solution, and culture temperature conditions. After the incubation, the germination rate and average germination length of the spores attached to each cover glass were measured by using an image analysis device (Visus image analysis, Ista-Video Test. Ltd., Russia). Figure 4 shows the germination rate of kelp spores under various culture conditions, Figure 5 shows the germination tube average length of the kelp spores under various culture conditions.

As shown in Figures 4 to 5, the kelp spores were smoothly germinated and germinated tube growth regardless of the amount of light irradiation. The culture temperature for germination and germination tube growth of kelp spores is in the range of 5 to 25 ° C., preferably 10 to 20 ° C., in particular, the culture temperature for germination tube growth is more preferably in the range of 15 to 20 ° C. Seemed. The pH of the culture medium for germination and germination tube growth of kelp spores was in the range of 5-9, preferably 6-9. In addition, the salinity of the culture medium was in the range of 15-55 ‰, preferably 25-45 ‰, and in particular, the pH of the culture medium for germination tube growth was more preferably in the range of 35-45 ‰.

2. 단일 금속류 독성 물질 용액의 수질 독성 평가2. Evaluation of Water Toxicity of Single Metal Toxic Solution

(1) Preparation of single metal toxic substance solution

Cadmium (initial concentration 20 mg / L), copper (initial concentration 0.4 mg / L), mercury (initial concentration 0.4 mg / L), nickel (initial concentration 20 mg / L), lead (initial concentration) 20 mg / L), diluted with half-water dilution using a solution of toxic substances containing zinc (initial concentration 20 mg / L) (salin concentration is 35 ‰) and OTT's artificial seawater (salin concentration is 35 ‰). Toxic solutions diluted to 50%, 25%, 12.5%, 6.25% of concentration were prepared and the water toxicity of these toxicant solutions was evaluated. As a control solution, OTT's artificial seawater (salin concentration: 35 ‰) containing no toxic substances was used.

(2) cultivation of kelp spores

Into a well of a 24-well plate (using a total of two 24-well plates), a control solution and a toxic substance solution were put into the well, and a cover glass with spores of Laminaria japonica was added thereto. I moved it. Thereafter, the cells were incubated at a temperature of 15 ° C. in a dark state for 24 hours.

(3) Measurement of germination rate and average length of germination tube of kelp spores

A total of 500 cultured spores were selected using an image analysis device (Visus image analysis, Ista-Video Test. Ltd., Russia) and the germination rates in the control solution and the toxic solution were determined by counting the number of germinated spores. In addition, the average length of the germination tube was determined by selecting 30 germinated spores, measuring the length from the beginning of the germination tube to the end of the germination tube of each spore and calculating their average value. At this time, the image of the spores cultured by adjusting the condenser provided in the optical microscope on the image analyzer was changed from white to black to observe the germination of the spores and the length of the germination tube.

Figure 6 is a graph showing the germination rate of kelp spores according to the concentration of toxic substances of metals, Figure 7 is a graph showing the germination tube average length of the kelp spores according to the concentration of toxic substances of metals. As shown in FIGS. 6 to 7, the germination rate of kelp spores or the germinal tube average length of the kelp spores showed a linear relationship with the concentration of metal toxic substances in a range.

(4) Determination of Water Toxicity of Single Metal Toxicity by Germination Rate of Kelp Spores and Germination Tube Average Length

Based on the germination rate and germination tube average length of the kelp spores shown in Figures 6 to 7 calculate the half effective concentration (EC ₅₀ ) to no effect concentration (NOEC) of a single metal-like toxic substance, based on the water quality evaluation criteria The sensitivity of the reaction to a single metal toxic substance was determined when the germination rate of the seaweed spores to the germinal tube length was determined. 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-like toxic substance based on the germination rate of germ spores to the germinal tube average length of water toxicity evaluation criteria.

As shown in Table 2, when the average length of germination tube was used as the standard for evaluating water toxicity, the response sensitivity to the single metal toxic substance was about 2 times higher than when the germination rate was used as the criteria for evaluating water toxicity. Very remarkable.

TABLE 2

Metal Toxic Substances	Germination rate standard		Germination tube average length
Metal Toxic Substances	NOEC (mg / L)	EC ₅₀ (mg / L)	NOEC (mg / L)	EC ₅₀ (mg / L)
CD	2.5	15.052	1.25	7.541
Cu	0.05	0.120	<0.025	0.081
Hg	0.025	0.041	<0.025	0.042
Ni	<1.25	2.009	<1.25	1.360
Pb	1.25	4.760	<1.25	4.429
Zn	1.25	6.049	<0.025	0.078

3. 단일 휘발성 유기화합물류 독성 물질 용액의 수질 독성 평가3. Evaluation of Water Toxicity of Single Volatile Organic Compound Toxic Solution

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

Single volatile organic compounds toxic substances, acetone (initial concentration 80 ㎖ / L), chloroform (initial concentration 1 ㎖ / L), dimethyl sulfate (initial concentration 40 ㎖ / L), ethyl alcohol (initial concentration 80 ㎖ / L) ), Methyl alcohol (initial concentration 80 ml / L), phenol (initial concentration 0.25 ml / L), toxic solution (salin concentration is 35 ‰) and OTT's artificial seawater (salin concentration is 35 ‰) Was diluted by half dilution method to prepare a toxic substance solution diluted to 50%, 25%, 12.5%, 6.25% of the initial concentration and evaluated the water toxicity of these toxic substance solutions. As a control solution, OTT's artificial seawater (salin concentration: 35 ‰) containing no toxic substances was used.

(2) cultivation of kelp spores

A total of 500 cultured spores were selected using an image analysis device (Visus image analysis, Ista-Video Test. Ltd., Russia) and the germination rates in the control solution and the toxic solution were determined by counting the number of germinated spores. In addition, the average length of the germination tube was determined by selecting 30 germinated spores, measuring the length from the beginning of the germination tube to the end of the germination tube of each spore and calculating their average value. At this time, the image of the spores cultured by adjusting the condenser provided in the optical microscope on the image analyzer was changed from white to black to observe whether the spores germinated and the length of the germination tube.

8 is a graph showing the germination rate of kelp spores according to the concentration of toxic substances of volatile organic compounds, Figure 9 is a graph showing the germination tube average length of the kelp spores according to the concentration of toxic substances of volatile organic compounds. As shown in FIGS. 8 to 9, the germination rate of kelp spores or the germinal tube average length of the kelp spores showed a linear relationship with the concentration of volatile organic compounds toxic substances in a certain range.

(4) Water Toxicity of Single Volatile Organic Compounds Toxic Substances by Germination Rate of Germ Spores and Germination Tube Average Length

Based on the germination rate and germination tube average length of the kelp spores shown in Figs. 8 to 9, half the effective concentration (EC ₅₀ ) to no effect concentration (NOEC) of a single volatile organic compound toxic substance is calculated, and based on the results The sensitivity of the response to a single volatile organic compound toxic substance was determined when the toxicity evaluation criteria were the germination rate of germ spores to germinal tube length. 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 a single volatile organic compound toxic substance based on the germination rate of germ spores to the germinal tube average length based on the evaluation of water toxicity.

As shown in Table 3, when the average length of germination tube was used as the standard for evaluating the water toxicity, the sensitivity of the reaction to a single volatile organic compound toxic substance was about 2 times higher than when the germination rate was used as the criteria for evaluating the water toxicity.

TABLE 3

Organic Compounds Toxic Substances	Germination rate standard		Germination tube average length
Organic Compounds Toxic Substances	NOEC (mg / L)	EC ₅₀ (mg / L)	NOEC (mg / L)	EC ₅₀ (mg / L)
Acetone	<5	10.402	<5	6.272
Chlororoform	0.125	0.617	<0.063	0.313
DMSO	2.5	21.705	2.5	11.649
Ethanol	<5	5.240	<5	2.974
Methanol	10	22.240	<5	13.161
Phenol	0.0625	0.187	<0.016	0.081

4. 염소를 독성 물질로 함유한 용액의 수질 독성 평가4. Evaluation of Water Toxicity of Solutions Containing Chlorine as Toxic Substance

The salt concentration of the clean stream influent and the effluent treated with a small amount of chlorine disinfectant was adjusted to 35 ‰. Thereafter, the influent solution and the effluent solution were used as the culture medium, and the kelp spores were cultured under the same culture conditions as those of the single metal toxic substance to the single volatile organic toxic substance. In addition, as a comparative example, the water toxicity of the influent solution and the effluent solution was measured using the Daphnia method. At this time, OTT's artificial seawater (salin concentration was 35 ‰) containing no toxic substances was used as a control solution. Table 4 shows the germination rate, germination tube average length, and daphnia survival rate of kelp spores cultured in influent and effluent solutions diluted to 50% of their initial concentration. And percentage value divided by the survival rate of Daphnia.

As shown in Table 4, the water fleas were killed in the effluent containing a small amount of chlorine disinfectant, and it was found that the Daphnia method using the water flea could not measure the water toxicity of a solution containing chlorine as a toxic substance. Can be. On the other hand, kelp spores are very stable in chlorine disinfectants, it can be seen that the water toxicity evaluation method of the present invention can be applied to measure the water toxicity of a solution containing chlorine as a toxic substance.

Table 4

Water sample classification	Daphnia Survival (%)	Germination rate of kelp spores (%)	Kelp germination tube average length growth rate (%)
Influent	100	100	100
Effluent	0	100	100

5. 파래의 포자 형성률, 다시마 포자의 발아율, 다시마 포자의 발아관 평균 길이를 기준으로 한 높은 염분 농도를 가진 수체 샘플의 수질 독성 평가5. Evaluation of Water Toxicity of Water Samples with High Saline Concentration Based on the Spore Formation Rate of Seaweed, Germination Rate of Kelp Spore, and Average Length of Germination Spore of Kelp Spore

Industrial wastewater influent (hereinafter referred to as influent) with an initial salt concentration of 40 ‰ and industrial wastewater effluent (hereinafter, referred to as effluent) with an initial salt concentration of 32 ‰ were purified. With OTT's artificial seawater (salin concentration is 35 ‰), the influent and discharge waters were diluted to 50%, 25%, 12.5%, and 6.25% of the original water concentration. Then, the kelp spores were cultured in the same manner as in the single metal toxic substance to the single volatile organic substance toxic substance using the raw water and the diluted raw water as the culture medium. As a comparative example, raw and diluted raw water were used as culture medium, and the coin-shaped perforated green leafs were cultured. (Cultivation condition: 100 μmol / m 2 · s of light irradiation, 12: 12h light cycle of light cycle, 15 ° C., incubation time 96h). The spore formation and release rate of the cultured perforated green leaves were measured by the area of color change. On the other hand, OTT's artificial seawater (salin concentration is 35 ‰) containing no toxic substances was used as a control solution. Table 5 shows the water toxicity results of inflow and discharge water measured on the basis of germination rate of germ spores, average length of germination tube, and rate of spore formation of green seed.

As shown in Table 5, it was determined that there was no water quality difference between the inflow and discharge water when the water toxicity evaluation method using the spore formation rate of the green sea was applied. On the other hand, the water toxicity evaluation method of the present invention using the germination rate of the kelp spores to the germination tube growth rate (average length) of the kelp spores is judged that there exists a certain degree of toxicity difference between the inflowed and discharged water, and more sensitive than the conventional technique. The discernment of

Table 5

Enemies	Raw water salinity (‰)	Raw water pH	Water Toxicity Evaluation Criteria	NOEC (%)	EC ₅₀ (%)	TU (toxic unit)
Influent	40	7.56	Kelp Spore Germination Rate	<6.25	32.50	3.08
			Kelp Spore Germination Tube Average Length	<6.25	21.24	4.71
			Green Spore Formation Rate	25	73.40	1.36
Discharge	32	7.77	Kelp Spore Germination Rate	6.25	70.87	1.41
			Kelp Spore Germination Tube Average Length	50	71.94	1.39
			Green Spore Formation Rate	25	75.00	1.33

※ In Table 5, NOEC, EC ₅₀ has units of "%" because the water sample contains a number of unknown toxic substances. For example, if the EC ₅₀ value of influent is 70%, Kelp spores cultured in samples diluted to 70% of initial concentration show spore germination rate or spore germination tube average length corresponding to 50% of the spore germination rate or spore germination tube average length of the control group.

The water toxicity assessment method or water toxicity assessment kit according to the present invention can be used to monitor whether seawater, river water, or lake is contaminated with toxic substances, and can quickly purify contaminated water bodies based on the evaluation results. will be.

Claims

Placing a water sample in a measuring container;

Injecting spores of algae belonging to the kelp in a measuring container containing a water sample;

Culturing the spores put into the measuring container; And

Measuring the germination tube growth rate of the spores germinated in the cultured spores; water quality toxicity evaluation method comprising a.
The method of claim 1, wherein the germination tube growth rate of the germinated spores is calculated as the germination tube average length of the germinated spores.
The method of claim 2, wherein the germination tube average length of the germinated spores is measured using a microscope equipped with a condenser for condensing light.
4. The method of claim 3, wherein the germination tube average length of the germinated spores is measured by changing the brightness of the cultured spore image by adjusting the condenser of the microscope.
The method of claim 1, wherein the water sample in the step of placing the water sample in the measuring vessel is adjusted to a salinity concentration of 25 ~ 45 ‰.
The water toxicity assessment according to claim 5, wherein the water sample adjusted to a salt concentration of 25 to 45 ‰ is diluted with artificial brine having a salt concentration of 25 to 45 ‰ and gradientd to at least five or more concentrations. Way.
The method of evaluating water toxicity according to claim 1, wherein the culturing the spores introduced into the measured container is incubated at a temperature of 10 to 20 ° C in a dark state.
The method according to any one of claims 1 to 7, wherein the seaweeds belonging to the kelp family are Ecklonia cava , Kjellmaniella crassifolia , Ecklonia stolonifera , Undaria pinnatifida , and Laminaria. japonica ) water quality toxicity evaluation method, characterized in that any one selected from the group consisting of.
The water sample according to any one of claims 1 to 7, wherein the water sample comprises cadmium (Cd; Cadmium), cobalt (Co; Cobalt), chromium (Cr; Chromium), copper (Cu; Copper), mercury (Hg; Mercury, Nickel, Pb, Lead, Zinc, Acetone, Acetone, Chloroform, Dimethyl sulfoxide, Ethyl alcohol And at least one toxic substance selected from the group consisting of formalin (formaldehyde), methyl alcohol (methanol), and phenol (Phenol).