KR20020056792A - Method of culturing domestic daphnia for environmental toxicity evaluation and method of measuring toxicity by use of same - Google Patents

Abstract

PURPOSE: Provided is a measurement method of environment toxicity by S.mixtus which uses the sensitive characteristics of S.mixtus against toxicity so that it is easily used as an environment monitor system for a big river like Han river or so on. CONSTITUTION: The measurement method of environment toxicity by S.mixtus is characterized by adding cultured S.mixtus to sample to check viable rate wherein the S.mixtus is cultured by collecting and culturing S.mixtus at 25deg.C for less than 24 hours to produce larvae. The culture is done at the concentration of one subject per more than 50ml of culture water including 320mg/l of CaCO3 and at less than 800 lux at light intensity for more than 18 hours per day.

Description

Cultivation method of domestic flea species for evaluation of environmental toxicity and method for measuring environmental toxicity using this flea species {METHOD OF CULTURING DOMESTIC DAPHNIA FOR ENVIRONMENTAL TOXICITY EVALUATION AND METHOD OF MEASURING TOXICITY BY USE OF SAME}

The present invention uses a method of culturing Simocephalus mixtus ( named witchcraft shimo flea in the country) and the S.mixtus which are inhabitants of lakes, rivers, swamps, etc. as indicator organisms of ecotoxicity assessment. To measure the toxicity.

Pollutants contain many environmental hazards (toxic) that can directly or indirectly affect organisms. Many experiments have been developed and used to measure them. However, it is not only impossible to measure and analyze both organic and inorganic pollutants, but also cannot accurately diagnose the effects of synergies between pollutants, and it is not economically recommended. In addition, various ecotoxicological concepts should be introduced into water quality management in order to minimize the influx of harmful chemicals contained in the waste water and to achieve the goal of water quality management such as preservation of human health and aquatic ecosystem.

As a method for the management of toxic substances in industrial wastewater, Canada has been evaluating the discharge of wastewater since 1970, and Denmark has been implementing this since 1974 and Finland since 1980. In addition, France uses toxicology test results when calculating emission charges, and Germany has been using various toxicology evaluation techniques in wastewater management since 1981. Ireland and Italy require toxicological testing of new installations when permitting new ones. In addition, countries that have legally regulated toxicity in wastewater, such as Sweden and Norway, or use toxicological data to regulate emissions, 15 Countries are reaching. In particular, the United States has been banning toxic emissions of toxic substances since 1972, and now all dischargers assess the toxicity of wastewater by the National Pollutant Discharge Elimination System (NPDES). To be discharged.

In addition, the early warning system using living organisms has been introduced and operated in order to monitor the inflow of harmful chemicals in the water system early. Especially, in the Rhine river system, a biological monitoring system is installed for the conservation of drinking water and ecosystem. The inflow is being monitored.

In Korea, water fleas are used to monitor the influx of harmful chemicals from the Han and Nakdong rivers at early stages, and toxicological tests are carried out for hazardous toxic substances. as a biological indicator and uses the Daphnia species dapeu California (Daphnia) imported exotics.

Republic of Korea Patent Application No. 98-58718 discloses an invertebrate yunchung acids (Rotifer) or daphnia (Daphnia magma as Ceriodaphnia dubia) toxicity that can be investigated using fresh water crustacean, such as fast and economical measurement of the waste water or the constant of dokseongdo The method is disclosed. This method makes it difficult for the larvae to live by adjusting the irradiation temperature of the incubation temperature, ultrasound or ultraviolet light to all the groups under the same conditions when the toxic substance and the invertebrate larvae come into contact with each other. Toxicity investigation method characterized in that to determine the toxic damage caused by the toxic substances on the basis of the mortality or movement of the larvae compared to the invertebrates injured by toxic substances compared to the group not injected with toxic substances to provide.

As mentioned above, Daphnia , the aforementioned imported species in the comprehensive environmental risk assessment of domestic hazardous toxic substances, is not only suitable for the domestic climate and the natural ecosystem, but also bears the economic cost of importing the imported species. There is a problem that if exposed to the natural environment by any path, there is a risk of disturbing the ecosystem.

In order to solve the above problems, the present invention provides a method for culturing Simocephalus mixtus , a water flea species inhabiting lakes, rivers and swamps in Korea, and a method for measuring toxicity and an environmental monitoring system using the same as an indicator organism. do.

1 is an optical micrograph of Simocephalus mixtus (country juvenile daphnia), one of the domestic flea species.

FIG. 2 is a domestic distribution diagram in which the magic shimoga flea shown in FIG. 1 is inhabited. FIG.

Figure 3 is an optical micrograph of the water flea taken at 40 times after incubation at 20 ℃ culture temperature.

Figure 4 is an optical micrograph of the water flea taken at 40 times after incubation at 25 ℃ culture temperature.

5a to 5e are micrographs showing the state of envelopment according to hardness.

6 is a graph showing the survival rate according to the hardness.

7 is a bar graph comparing the heavy metal toxicity sensitivity of water flea according to the assay culture temperature.

8 is a schematic diagram of a toxicity experiment using daphnia.

9a to 9f are bar graphs showing the results of Upo Wetland Sediment Toxicity test using daphnia.

10a and 10b are graphs showing the results of toxicity tests by depth of Upo Wetland sediment using water fleas.

Korean fleas used in the present invention have a form as shown in Figure 1, the scientific name is Simocephalus mixtus , also known as witch Shimo Shim Daphnia. This S.mixtus species is classified as phylogenetic and belongs to Arthropoda, Crustacea, Cladocera and Daphnidae.

This water flea is inhabited throughout the country as shown in Figure 2 [2000, Yoon Sung-myeong · Kim Won], Nakdong River, Upo Wetland, Toppyeongcheon, nature learning centers, farms, rice fields, etc. It can be easily collected by those skilled in the art.

Means for evaluating the effects on living organisms in chronic toxicity experiments are the rate of nesting or fertility. Therefore, in order to be useful for the toxicity test, the optimum culture conditions were obtained to maximize the incubation rate and fertility rate of the collected domestic fleas, and the sensitivity of heavy metal toxicity according to the culture conditions was confirmed. In addition, by comparing the toxicity sensitivity between the domestic species of the present invention and the conventional foreign species was confirmed the superiority of the domestic flea species of the present invention.

Example 1

Culture Effect According to Culture Condition

1) Effect of Culture on Temperature

In order to determine whether the incubation temperature affects the incubation rate or fertility rate, S.mixtus was incubated at 20 ° C and 25 ° C, respectively, and then observed under a 40x optical microscope. The result is as shown in FIG. 3 and FIG.

Comparing the photographs of the individuals incubated for the same time, FIG. 3, that is, the individuals incubated at 20 ° C. are not only small in size, but do not have eggs. Can be. Juvenile fleas were found to be effective in culturing in view of the higher fertility rate in better environment.

2) Culture effect according to food type

To investigate the effect of culture on different food types, Chlorella and Scenedesmus species with the same total organic carbon (TOC) concentration were fed. Each of them was cultured in three repetitions, and the initial number of oviparation, the number of births and the number of births after 10 days were observed and compared. The results are shown in Table 1 below.

Culture status by food type Chlorella sp. Scenedesmus sp. Primary Secondary 3rd Primary Secondary 3rd 1) First egg day (Sun) 4 4 4 4 4 4 2) First childbirth (mari) 8 6 6 8 8 6 3) The number of the births after 10 days (mari) 52 40 45 44 46 33

As can be seen from the results of Table 1, in the 95% confidence intervals, the results of the t-test test showed no difference in terms of 1), 2), and 3). Therefore, it can be seen that there is no difference in the culture effect according to the type of food.

3) Optimum culture conditions according to hardness

The results were reviewed based on the survival rate of S.mixtus and the number of neonates produced by the individual. As a result, the species incubated at a hardness of 120-140 mg / L CaCO3 had the best degree of encapsulation. The nesting state according to the hardness is shown in FIGS. 5A to 5E. As a result of the 21-day chronic test, as shown in FIG. 6, the survival rate was 40% in the very soft water and the survival rate was 60% in the very hard water. On the other hand, in soft water, moderately hard water, proposed hard water, and hard water, the survival rate was 100%.

4) Other culture conditions

Other culture conditions were obtained based on the optimum culture conditions of water fleas known in the art, such as the following optimum conditions for S.mixtus .

Culture Water: 80-Contains CaCO3 320mg / L

Feeding amount: 0.8mg / L / 1 object / day

Culture chamber size: 1 individual / 50 ml or more

Light cycle: light illumination for 18 hours / day

Light intensity: below 800 lux

Using the larvae incubated for less than 24 hours S.mixtus according to the culture conditions, the sensitivity to heavy metal toxicity of water fleas described in the following Example 2 was tested. The reason why the larvae were used as the target organisms of the toxicity experiment was that the younger the individuals, the more susceptible to the toxic substances.

Example 2

Heavy Metal Toxicity Sensitivity Test with Analytical Culture Temperature

To determine whether the heavy metal toxicity sensitivity of S.mixtus incubated for less than 24 hours changed with the temperature of the toxicity test, the larvae were tested at 20 ° C and 25 ° C, respectively.

Specifically, the heavy metals used were Cu, Cd, and Zn, and each was used at a concentration of 500 ppb. Ten larvae were injected into 100 ml of each sample, and then cultured at 20 ° C. and 25 ° C., respectively, and the number of surviving individuals after 6 hours of Cu, 1 hour 30 minutes, and 24 hours of Zn was counted.

The results are shown in FIG. As shown in FIG. 7, toxic larvae at 25 ° C. were killed at least 50%, while toxic larvae at 20 ° C. survived almost 80%. Therefore, the temperature was found to be a factor affecting the sensitivity of the toxicity test, it was confirmed that 25 ℃ is a favorable condition for the culture effect and sensitivity.

Example 3

S.mixtus Sensitivity measurement

In order to evaluate the sensitivity level of the toxicity test of S.mixtus , the toxicity test was performed using the existing D.magna and C.dubia together with the S.mixtus of the present invention. To test the toxicity, samples were taken from the tributaries of the Nakdong River, in front of the farm (Changnyeong Upo Swamp), Mokpo + Upo (Changnyeong Upo Swamp), in front of the rice fields (Changnyeong Upo Swamp), the Inpyeongcheon Inflow and Nature Learning Center (Changnyeong Upo Swamp). After homogenization, the effluent and the pore water were extracted, and each of the extracted samples was subjected to toxicity experiments.

The pore water sample was centrifuged at 250 ° C for 250 minutes at 8000 rpm for 45 minutes, the supernatant was filtered through a filter paper, and then the pH and dissolved oxygen (DO) were adjusted. After diluting at a rate of 30 rpm for 30 minutes with a jar-test at 300rpm, the mixture was allowed to stand overnight, and only the supernatant was collected and centrifuged at 10,000rpm for 15 minutes. This sample was also used to adjust the pH and DO.

Culturing a S.mixtus, D.magna C.dubia and cultured as in Example 1 to those for toxicity test sample under optimal toxicity test conditions described in Example 2 were observed for each of the LT50.

A schematic procedure of this experimental method is shown schematically in FIG. 8, and the test results are as shown in FIGS. 9A to 9B.

In addition, Mokpo + Upo Wetland and Nature Learning Center were sampled by the depth of the sediment and tested the toxicity sensitivity of the three water flea species to each sample. The results are shown in FIGS. 10A and 10B.

Finally, the heavy metal toxicity sensitivity exhibited by the three daphnia species was assessed by 48hr-LC50 known in the art. The evaluation results are shown in Table 2.

Exotic species Daphnia D.magna C.dubia and heavy metal toxicity and sensitivity tests of domestic species, habitat S.mixtus Daphnia type Metal (μg / L) Cu Zn CD D. magna 97 (118) 59 (68) 47 (54) C. dubiaa) (110) (76) (27) S. mixtus 44 56 35 (): 48hr-LC50m, D.I. Mount et. Al (1984), cited a): Cited by Margaret W. Toussaint et. Al (1995).

As can be seen Fig. 9a to 9f, with reference to FIG. 10a and 10b and Table 2, it was confirmed that the local template species S.mixtus the most sensitive as compared to the non-native D.magna and C.dubia.

Simocephalus mixtus , a juvenile shimoda flea species inhabiting the whole country, does not need special conditions for cultivation and toxicity evaluation other than temperature. In particular, because it is highly sensitive and already adapts to the domestic environment, the degree of adaptation or reaction to various pollutants is faster than that of the imported species, and the error of experimental results can be reduced as much as possible. Exposure is unlikely to disrupt ecosystems, unlike exotic species.

In addition, the present invention in the comprehensive environmental risk assessment of the pollutants by replacing the organisms that depended on the existing foreign imported species with domestic species to reduce the foreign currency caused by the import of the foreign species, domestic paper species such as purchase and culture An environmental monitoring system can be provided that reduces the economic burden. In particular, by standardizing the measurement of the toxicity of water fleas in accordance with the natural ecosystem of Korea by using organisms suitable for the domestic climate, it can be very helpful in setting the criteria of the national environmental risk assessment method.

As mentioned above, although this invention was demonstrated with reference to the preferred embodiment of this invention, those of ordinary skill in the art should understand this invention in the range which does not deviate from the idea and the scope of the invention as described in the following claims. It will be appreciated that various modifications and changes can be made.

Claims (5)

A method of cultivating daphnia species for the evaluation of environmental toxicity, characterized by harvesting Simocephalus mixtus which is a domestic daphnia species and incubating at 25 ° C. for less than 24 hours. The method of cultivating daphnia species according to claim 1, wherein the culturing is carried out at a concentration of one individual per 50 ml or more of the culture water in the culture water containing 320 mg / L of CaCO3. The method of culturing daphnia species according to claim 1 or 2, wherein the light intensity at the time of culturing is 800 lux or less and is illuminated for 18 hours of the day. A method for measuring environmental toxicity, comprising adding Simocephalus mixtus cultured by the method of claim 1 to a sample and checking the survival rate. 5. The method of claim 4, wherein the Simocephalus mixtus by exposure to heavy metals is also measured how environmental toxicity, characterized in that to evaluate the toxicity sensitivity to 48hr-LC50.