KR101136040B1 - Method for evaluating aquatic ecotoxicity using the root growth length or the root growth rate of lettuce seed - Google Patents

Abstract

PURPOSE: A method for evaluating water toxicity is provided to ensure sensitive and accurate evaluation and to remarkably shorten evaluation period. CONSTITUTION: A method for evaluating water toxicity comprises: a step of preparing water samples including four or more of dilution water which has different culture liquid, original water, and dilution ratio without toxic material; a step of adding the water samples to a culture device having at least six wells; a step of adding lettuce seeds to the culture device; a step of culturing the seed; and a step of measuring root length or root growth rate.

Description

Method for evaluating aquatic ecotoxicity using the root growth length or the root growth rate of lettuce seed}

The present invention relates to a method for evaluating water toxicity, and more particularly, to a method for evaluating water toxicity using root growth length or root length growth rate of lettuce seeds.

As the industry advances, the toxic and harmful substances contained in various water bodies also increase, causing damage that was not possible in the past. In other words, due to environmental pollution such as soil pollution, water pollution, etc., the ecosystem is gradually destroyed and affects the human living environment. Accordingly, the world is trying to reduce environmental pollution such as water pollution by further strengthening environmental regulations on a national level. However, related laws do not keep up with the speed of increase of factories due to industrialization. Therefore, in accordance with the related laws, a method of simply examining environmental pollution such as water pollution has been continuously studied, but satisfactory results have not been obtained.

Bioassay is the most widely used method of evaluating ecotoxicity, and it is a method of evaluating the toxicity by measuring biological response factors such as death, growth, hatching and fertilization by exposing the organism to harmful substances. . For example, when a harmful substance is exposed to the organism in an aqueous solution, the organism is killed or the growth is judged to be harmful if the growth shows a large difference compared to the control (experimental group without addition of harmful substances). This method is based on the toxicity assessment of individual substances by conventional chemical analysis (expensive analytical equipment and highly trained analytical personnel, long analysis times and unknown constituents of toxic substances). It is difficult to analyze, and when moving the sample over a long distance, there is a problem that the reaction in the sample may cause a change in composition and concentration. According to this method, the half lethal concentration (LC ₅₀ ) and the half effective concentration (EC ₅₀ ) are calculated and the relative quality of the environment and the toxicity of the substance are compared according to the comparison of these values.

The organisms used in the bioassay are selected based on the availability of the species, the sensitivity to toxic substances and the consistency of the response, resistance to environmental factors and ecological significance. The use of fish, invertebrates or protozoa requires a specialist's expertise and expensive equipment, and the sensitivity varies depending on the toxic substances. There is a problem that countermeasures are unknown. Currently, living organisms mainly used in the United States, Europe, etc. are rotifers, crustaceans, fish, benthic, monopods and luminescent microorganisms. Although methods for diagnosing water toxicity using living organisms have been developed to compensate for the shortcomings inherent in the results of methods using physicochemical devices, ecotoxicity has been registered internationally in ISO and OECD. Evaluation techniques absolutely require the emergence of better techniques due to the complexity of the procedure, instability of sample acquisition, low sensitivity, constraints on the area of biologic distribution used, high cost, and difficulty in cultivating, supplying and maintaining test organisms. It is a situation.

On the other hand, lettuce ( Lactuca) among the organisms used for the evaluation of ecotoxicity sativa ) is widely used for the edible cultivation of the bellflower, asteraceae, and is a convenient paper for the study of physiological responses to various environmental factors because it is possible to obtain abundant samples and to be easily cultured and manipulated in the laboratory. In addition, the lettuce has the advantage that it is easy to obtain the material and can buy a large amount of material at a low price because it is easy to purchase various varieties of seedlings in general commercial gardens and flower gardens. In addition, the storage and maintenance of the sample is convenient, making it easy to carry out the test, and the test can be performed in a small space, which has the advantage of simultaneously measuring a large number of samples.

The US Environmental Protection Agency (US EPA) lettuce bioassay method of the conventional ecological toxicity evaluation method using lettuce lettuce consists of measuring germination and root growth of lettuce seeds, as shown in Figure 1 Petri dishes (petridish) A filter paper (usually Wattman) is placed inside and wetted with a culture solution to induce the germination of lettuce seeds and evaluate the ecotoxicity by the germination rate or the degree of root growth. However, the conventional method of evaluating ecotoxicity using lettuce has a problem in that the toxicity of the water sample is reduced by the filter paper, which causes the problem of being underestimated than the actual toxicity. In addition, the germination and growth time of the lettuce seed takes about 5 days there is a problem that the rapid evaluation is not made. In addition, as shown in FIG. 2, the US Environmental Protection Agency's lettuce bioassay test has a limit in measuring accurate root growth because the roots of lettuce seeds grow in a curved shape.

The present invention was derived to solve the conventional problems, the present invention uses the root growth length or root length growth rate of lettuce seeds as an indicator of water toxicity evaluation, increase the number of experiment group repeats in one experiment, lettuce seeds By establishing the optimal culture conditions for root length growth of the cultivation, it increases the objectivity of the experiment, reduces the toxic substances during the evaluation process, prevents the underestimation of water toxicity, has excellent sensitivity and accuracy, and significantly shortens the toxicity evaluation period. The aim is to provide a method for evaluating water toxicity that can be used.

In order to solve the above object of the present invention, the present invention comprises the steps of: (a) preparing a sample of water containing no toxic substance, raw water, at least four dilution raw water of different dilution multiples; (b) placing the water sample in an incubator having at least six wells; (c) injecting lettuce seeds into an incubator containing a water sample; (d) culturing the lettuce seeds introduced into the incubator; And (e) measuring the root growth length or root length growth rate of the lettuce seed culture is completed; provides a method for evaluating water toxicity.

At this time, the dilution raw water of step (a) is preferably characterized in that it is prepared by a half-water dilution method.

In addition, the water sample of step (b) is preferably characterized in that the salt concentration is adjusted to 0 ~ 5 ‰, pH is adjusted to 5 ~ 9.

In addition, the culture conditions of step (d) is characterized in that the light irradiation amount is 0 ~ 150 μmol photon / ㎡ · s and the culture temperature is 15 ~ 25 ℃.

In addition, the water toxicity evaluation method according to the invention preferably further comprises the step of culturing the lettuce seeds in a standard toxic solution prior to step (a) to evaluate the condition of the lettuce seeds, wherein the standard toxic solution is silver (Ag Silver, aluminum, arsenic (As; Arsenic), cadmium (Cd; Cadmium), cobalt (Co; Cobalt), chromium (Cr; Chromium), copper (Cu; Copper), iron, mercury (Hg; Mercury), One metal toxic substance selected from the group consisting of nickel (Ni), lead, and zinc is dissolved in distilled water.

In the method of evaluating water toxicity according to the present invention, since the raw water dilution obtained by diluting control, raw water, and raw water to four or more different concentrations is used as a culture medium for lettuce seeds in a plate incubator having at least six wells. In evaluating water toxicity, the number of replicates in the experimental group can be increased to increase the objectivity of the experiment. In addition, because lettuce seeds are suspended in culture without the use of filter paper, toxic substances are reduced during the evaluation process, which prevents underestimation of water toxicity. Growth length can be measured. In addition, since the cultivation is carried out under culture conditions that are optimal for root length growth of lettuce seeds, the incubation time is only about two days, so that the evaluation time can be significantly shortened.

Figure 1 shows the configuration of the lettuce bioassay test of the US Environmental Protection Agency (US EPA), Figure 2 shows the root growth characteristics of lettuce seeds cultured by the lettuce bioassay of the US Environmental Protection Agency (US EPA) under a microscope It is a video.
Figure 3 shows the configuration of the water toxicity evaluation method according to the present invention, Figure 4 is an image of the root growth characteristics of lettuce seeds cultured by the water toxicity evaluation method according to the present invention under a microscope.
5 is a graph showing the germinated root growth length of lettuce seeds cultured in various culture conditions.
FIG. 6 is a microscope of root growth characteristics of lettuce seeds cultured for 48 hours by toxic aqueous solution containing copper (Cu) as a single toxic substance (initial copper concentration is 1 mg / L) and toxic aqueous solution diluted by half dilution method. Observed image (culture condition is 20 ℃, dark state).
7 is a graph showing the root length growth rate of lettuce seeds according to the concentration of toxic substances in metals.

The present invention relates to a method for evaluating water toxicity, which is simple, has a short discrimination time, and accurately evaluates the toxicity exerted by a toxic substance on a water sample. The method of evaluating water toxicity according to the present invention does not include a toxic substance. Preparing a water sample consisting of at least four dilution raw water having different culture solution, raw water and dilution drainage; Placing the water sample in an incubator having at least six wells; Injecting lettuce seeds into an incubator containing a water sample; Culturing the lettuce seeds added to the incubator; And measuring the root growth length or the root length growth rate of the cultured lettuce seeds. 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 first aqueous sample (hereinafter referred to as raw water) is preferably diluted to at least four different concentrations using a liquid that does not contain toxic substances such as distilled water. It is used as a culture medium for cultivating lettuce seeds. The dilution method used to prepare the dilution raw water is not very limited, for example half dilution method [100% (raw water itself), 50% (diluted to 1/2 concentration of raw water), 25% (1 / of raw water) 4 dilute), 12.5% (diluted to 1 / 8th the concentration of raw water), 6.25% (diluted to 1 / 16th concentration of raw water)].

The culture medium that does not contain the toxic substance constituting the control group is not particularly limited as long as it is compatible with the lettuce seeds without inhibiting the germination and root growth of the lettuce seeds, specifically, distilled water.

On the other hand, the aqueous sample consisting of the control, raw water and dilute raw water is preferably adjusted to a salinity of 0 ~ 5 ‰, pH is adjusted to 5 ~ 9 before cultivating lettuce seeds. Germination and root growth of lettuce seeds are facilitated at the salt concentration and pH.

In the present invention, in the case of cultivating lettuce seeds with a salt sample (consisting of control, raw water, and 4 or more dilute raw waters) of which the salt concentration and pH are adjusted, the root growth of the lettuce seeds according to the dilution concentration of the raw water Length or root length growth rate can be obtained and based on this, when assessing the water toxicity of a water sample, the exact half effective concentration (50% less than the root growth length or root length growth rate of lettuce seeds in the control group without toxic substances). Concentrations of toxic substances with a defined root growth length or root length growth rate, 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 (toxic substance) Significant difference from the root growth length or root length growth rate of lettuce seeds in the control group In the case of single toxins it appears in a specific concentration, and the case of the raw image shown in dilution ratio as the concentration of toxic substances to maintain a level; can be evaluated in the No Observed Effect Concentration, NOEC).

Incubator

In the water toxicity evaluation method according to the invention it is preferred that the incubator has at least six wells. Figure 3 shows the configuration of the water toxicity evaluation method according to the invention and Figure 4 is an image of the root growth characteristics of the lettuce seed cultured by the water toxicity evaluation method according to the present invention under a microscope. As shown in FIG. 3, when a 6-well plate incubator is used as the incubator, one well is added with a culture medium containing no toxic substance as a control to incubate twin seeds, and the remaining five wells are diluted with raw water and raw water. A water sample can be added to cultivate twin seeds. In the case of using an incubator having six or more wells, the number of experiment groups can be increased to increase the objectivity of the experiment, and at the same time, the filter paper does not exist, thereby preventing the reduction of toxic substances by the filter paper. In addition, the floating culture is possible, as shown in Figure 4, the root growth of the lettuce seed is made of a straight shape rather than bending. In addition, the degree of germination and root growth of lettuce seeds increases than when culturing on filter paper during suspension culture, so that the root growth length is more than 10 mm after 48 hours and can be visually confirmed.

Lettuce seeds

The present invention uses the root growth length or root length growth rate of the lettuce seeds, more specifically lettuce seeds as a biomarker for the evaluation of water toxicity, lettuce seeds are contaminated or the degree of germination in the water containing the toxic source And root growth is poor. Lettuce seeds are preferably healthy seeds with light gray surface, fine wrinkles, and a long width of 2.5 mm or more. If the lettuce seeds are unhealthy, it may be difficult to assess significant water toxicity due to the lack of germination and root length growth.

Cultivation of Lettuce Seeds

In order to maximize the germination and root growth by culturing the lettuce seeds, the light irradiation amount in the culture is preferably 0 to 150 μmol photon / m 2 s, more preferably 10 to 150 μmol photon / m 2 s Incubation temperature is 15-25 degreeC, More preferably, it is 20-25 degreeC. In addition, the pH of the culture medium is 5-9, more preferably 6-9 and the salt concentration is preferably 0-5 ‰. In the water toxicity evaluation method according to the present invention, the incubation time of the lettuce seed is about 48 hours (2 days), which is 72 hours shorter than that of the US water agency lettuce water toxicity evaluation method.

Determination of Root Growth Length or Root Length Growth Rate of Lettuce Seeds

After the incubation is completed, the root growth length of the lettuce seed may be measured visually using a ruler, or may be measured using an image analysis device having an image capture such as a camera. Root length growth rate from the measured root growth length can be calculated by the following equation (1).

Where A represents the root growth length of the lettuce seed in the control group (without the toxin source), and B represents the root growth length of the lettuce seed in the experimental group (water samples containing the toxin source).

Assessment of Water Toxicity

Water quality toxicity of the water sample may be assessed using the root growth length of the lettuce seed itself or may be evaluated using the root length growth rate.

The method using the root growth length itself is as follows.

Where A represents the root growth length of the lettuce seed in the control group (without the toxin source), and B represents the root growth length of the lettuce seed in the experimental group (water samples containing the toxin source).

In addition, water toxicity was assessed by comparing the root growth length or root length growth rate of the lettuce seeds cultured in the water samples of raw and diluted raw waters to the root growth length or root length growth rate of the lettuce seeds cultured in the control group containing no toxic substances. Specifically, it is expressed as a value of Half maximal effective concentration (EC ₅₀ ) to No Observed Effect Concentration (NOEC).

Half maximal effective concentration (EC ₅₀ ) means the concentration of a water sample effective to reduce root growth length or root length growth rate by 50% less than root growth length or root length growth rate in a control free of toxic substances. As an aquatic sample containing a single toxic substance, it is expressed as a specific concentration of a single toxic substance, and for an unknown raw water containing a plurality of toxic substances, it is expressed as the dilution rate of the raw water. Unaffected concentration refers to the concentration of a water sample effective to maintain root growth length or root length growth rate at a level that is not significantly different from root growth length or root length growth rate in a control group that does not contain toxic substances. Like concentrations, water samples containing a single toxic substance are indicated at a certain concentration, and for unknown raw water containing a large number of toxic substances, the dilution rate is indicated. 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

Toxic substances that can be diagnosed by the water toxicity evaluation method of the present invention include silver (Ag; Silver), aluminum, arsenic (As; Arsenic), cadmium (Cd; Cadmium), cobalt (Co; Cobalt), chromium (Cr; Chromium), Heavy metals such as copper (Cu), iron, mercury (Hg; Mercury), nickel (Ni), lead, and zinc.

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 quality test kit according to the present invention comprises lettuce seeds; An incubator having at least six wells as an incubator for injecting a water sample and lettuce seed and for cultivating twin seeds; And dilution water to dilute the water sample; It includes. In addition, the water toxicity evaluation kit according to the present invention is silver (Ag; Silver), aluminum, arsenic (As; Arsenic), cadmium (Cd; Cadmium), cobalt (Co; Cobalt), chromium (Cr; Chromium), copper ( A standard toxic substance solution prepared by dissolving one metal toxic substance selected from the group consisting of Cu; Copper), iron, Hg; Mercury, Ni; Nickel, lead, and zinc in dilution water is added. Standard toxicant solutions may be used to test the condition of lettuce seeds, ie reaction sensitivity to toxicants, prior to assessing the water toxicity of a water sample. Specifically, if the standard toxic substance solution according to the present invention is a solution containing copper as a toxic substance, the water toxicity evaluation kit by obtaining data on the root growth length or root length growth rate of the lettuce seeds in various copper solutions and the control group in advance Can be provided with The user may determine whether the lettuce seeds will normally respond to toxic substances by measuring the root growth length or root length growth rate of the lettuce seeds against the copper solution and comparing them with previously provided data, If more than 80% of the sensitivity of the response is shown, the assessment of water toxicity on unknown water samples can be performed.

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.

1. Establish Optimal Culture Conditions of Lettuce Seeds

The lettuce seeds, which are commercialized and sold in general gardens and seedlings, have been purchased for 2-3 years and distilled water was used as a culture medium.

The culture solution was put in each well of a 6-well plate incubator, and lettuce seeds were added thereto, followed by incubation under various light doses, pH of the culture solution, salt concentration of the culture solution, and culture temperature conditions. After 48 hours of incubation, the lettuce seeds were placed on a slide glass and the root growth length of the lettuce seeds was measured by using an image analysis apparatus (Visus image analysis, Ista-Video Test. Ltd., Russia) and a Motic image program.

Figure 5 shows the root growth length of lettuce seeds in various culture conditions. Looking at the optimum culture conditions, the culture medium had a salt concentration of 0 to 5 ‰, pH of 5 to 9, preferably 6 to 9, the culture conditions of the light irradiation amount of 0 ~ 150 μmol photon / ㎡? S, preferably It was 10-150 micromol photon / m <2> * s, and the culture temperature was 15-25 degreeC, Preferably it was 20-25 degreeC.

2. Evaluation of Water Toxicity of Single Metal Toxic Solution

(1) Preparation of single metal toxic substance solution

As a single metal toxic substance, distilled water contains silver (Ag; Silver), aluminum, arsenic (As; Arsenic), cadmium (Cd; Cadmium), cobalt (Co; Cobalt), chromium (Cr; Chromium), copper (Cu; Copper) Cadmium, copper, lead and zinc mother liquor are added to iron, mercury (Hg; Mercury), nickel (Ni), lead, and zinc, to prepare raw water containing a single metal toxic substance, and dilute the raw water by half dilution. Toxicity dilution raw water diluted to 50%, 25%, 12.5%, 6.25% of the initial concentration was prepared to evaluate their water toxicity. Distilled water was used as a control solution. The salinity of raw water, dilute raw water and control was 0 ‰ and pH was adjusted to 7.0.

(2) cultivation of lettuce seeds

Raw water, dilution water, and control were placed in a 6-well plate incubator and lettuce seeds were added thereto, followed by incubation for 48 hours. At this time, the culture conditions were 0 μmol photon / m 2 s (cancer culture) and the temperature was 20 ° C.

(3) Determination of Root Growth Length and Root Length Growth Rate of Lettuce Seeds

After 48 hours of incubation, the lettuce seeds were placed on a slide glass and the root growth length of the lettuce seeds was measured by using an image analysis apparatus (Visus image analysis, Ista-Video Test. Ltd., Russia) and a Motic image program.

FIG. 6 is a microscope of root growth characteristics of lettuce seeds cultured for 48 hours by toxic aqueous solution containing copper (Cu) as a single toxic substance (initial copper concentration is 1 mg / L) and toxic aqueous solution diluted by half dilution method. Observed image (culture condition is 20 ℃, dark state). 7 is a graph showing the root length growth rate of lettuce seeds according to the concentration of toxic substances in metals.

From the results shown in FIGS. 6 to 7, a half effective concentration (EC ₅₀ ) to no effect concentration (NOEC) of the toxic substance may be calculated through various statistical techniques such as a linear interpolation method.

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 Figure 7, the% control of the vertical axis represents the root length growth rate calculated by Equation (1).

Claims (6)

(a) preparing a water sample consisting of at least four dilution raw water with different culture medium, raw water, and dilution multiples free of toxic substances;
(b) placing a water sample in each well of an incubator having at least six wells;
(c) injecting lettuce seeds into each well of an incubator containing a water sample;
(d) culturing the lettuce seeds added to each well of the incubator; And
(e) measuring the root growth length or root length growth rate of the incubated lettuce seed; and
The dilution raw water is a dilution of raw water with distilled water,
The toxic substance is silver (Ag), aluminum, arsenic (As; Arsenic), cadmium (Cd; Cadmium), cobalt (Co; Cobalt), chromium (Cr; Chromium), copper (Cu; Copper), iron, Mercury (Hg; Mercury), nickel (Ni; Nickel), lead, and water quality toxicity method characterized in that at least one metal selected from the group consisting of zinc.
The method of evaluating water toxicity according to claim 1, wherein the dilution raw water of step (a) is prepared by half dilution method.
The method of claim 1, wherein the culture medium, raw water, and at least four or more dilution raw water having different dilution multiples, each containing no toxic substance constituting the water sample of step (a), are respectively adjusted to a salt concentration of 0 to 5 ‰. And, pH is adjusted to 5-9.
The method of evaluating water toxicity according to claim 1, wherein the culture conditions of step (d) have a light irradiation amount of 0 to 150 μmol photon / m 2 · s and a culture temperature of 15 to 25 ° C.
The method of any one of claims 1 to 4, further comprising the step of culturing the lettuce seeds in a standard toxic solution prior to step (a) to assess the condition of the lettuce seeds. .
The method of claim 5, wherein the standard toxic solution is silver (Ag; Silver), aluminum, arsenic (As; Arsenic), cadmium (Cd; Cadmium), cobalt (Co; Cobalt), chromium (Cr; Chromium), copper ( Water toxicity, characterized in that one metal toxic substance selected from the group consisting of Cu; copper), iron, mercury (Hg; Mercury), nickel (Ni; Nickel), lead, and zinc is dissolved in distilled water Assessment Methods.

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