KR102272329B1 - Method for diagnosing pollution of marine sediment using floating egg of fish - Google Patents

Abstract

The present invention comprises the steps of: (a) placing a sample containing the sediment into a test chamber; (b) injecting seawater with a controlled salinity concentration into the test chamber, leaving the sediment and seawater in a parallel state, and checking whether the salinity concentration of the seawater in the test chamber is the same; (c) placing the fish eggs into the test chamber; (d) separating the hatched fish individual from the test chamber after the exposure time has elapsed; And (e) it provides a method for diagnosing sediment contamination using floating fish eggs, comprising the step of counting surviving individuals from among the separated fish individuals.
The method for diagnosing sediment contamination using floating fish eggs of the present invention can be usefully used as a technology capable of diagnosing whether sediment is contaminated, inexpensively, easily and conveniently in the field of marine environmental pollution.

Description

Method for diagnosing pollution of marine sediment using floating egg of fish

The present invention relates to a method for diagnosing sediment contamination using floating fish eggs, and more particularly, to a method for diagnosing contamination of marine sediments easily and conveniently using fertilized halibut eggs that can control the water level according to salt concentration.

As the global population increases, food consumption increases and the use of pesticides is rapidly increasing to increase agricultural productivity. On the other hand, due to the construction of industrial complexes, contaminated wastewater from the land flows into rivers, rivers and seas excessively. It is becoming. Such marine pollution causes pollution of coastal fisheries, which is also a problem in the supply of fishery products.

Various methods of research are being conducted to evaluate and diagnose the pollution of seawater and sediments, which are getting serious day by day. In general, quantitative evaluation of the concentration of pollutants is performed, but the analysis method is quite complicated and expensive and expensive. There was a disadvantage in that it took a long time for analysis. Another evaluation method, the biological evaluation (toxicological evaluation) method, can present important data to evaluate the risk in the real environment by measuring the biological effect, and its importance is being emphasized in recent years.

Biological evaluation using sediment is performed to determine the biological effects that contaminants in sediment can cause. In fact, changes in organism motility, mortality, and reproduction rate in sediment exposure tests using various benthic organisms such as midges and bivalves. (Phelps HL. 1989. Bull Environ Contam Toxicol. 43(6): 838-845, Bat L, et al., 1998. J Exp Mar Biol Ecol. 226(2): 217-239). In particular, recently, studies using molecular biomarkers to diagnose biological effects at a more sensitive level than individuals have been conducted.

However, the use of molecular biomarkers for sediment contamination evaluation is different from biomarkers in living organisms living in the field, so it is only performed in exposure experiments using large benthic animals, which are organisms that can be observed by exposing them to site sediment. In the case of living organisms, there are difficulties in maintaining and securing the living organisms in the laboratory. In addition, the evaluation method directly exposed to sediment cannot exclude the effects of particles that can be food or the biological effects of exposure to pollutants through the feeding route, and there are restrictions on the experimental species, so various experiments are required for accurate evaluation. and collection of related data is required.

Therefore, it is required to develop a technology capable of early evaluation of contamination of marine sediments and of inexpensive, easy and convenient diagnosis.

Korean Patent Registration No. 10-1537810 (2015.07.13.) Republic of Korea Patent Registration No. 10-0681410 (2007.02.05.)

Eun Ji Won et al., J. Environ. Impact Assessment. (2017) 26(3): 207~216 Z. Chial Belgis et al., Chemosphere 52 (2003) 95-101

While the inventors of the present invention were studying a method for diagnosing the contamination of marine sediments easily and conveniently, the difference in salinity of seawater by using floating eggs that float on water because of their light specific gravity as oil droplets in eggs among fertilized eggs of fish It was found that the toxicity according to the degree of dissolution of the sediment could be checked by locating the floating egg at a distance close to the sediment by adjusting the position of the water depth according to the method and exposing it for a certain period of time.

Accordingly, an object of the present invention is to provide a method for diagnosing sediment contamination using floating fish eggs.

According to one aspect of the present invention, there is provided a method comprising: (a) placing a sample comprising a sediment into a test chamber; (b) injecting seawater with a controlled salinity concentration into the test chamber, leaving the sediment and seawater in a parallel state, and checking whether the salinity concentration of the seawater in the test chamber is the same; (c) placing the fish eggs into the test chamber; (d) separating the hatched fish individual from the test chamber after the exposure time has elapsed; And (e) there is provided a method for diagnosing contamination of sediments using floating fish eggs, comprising the step of counting surviving individuals from among the separated fish individuals.

In one embodiment, the sediment of step (a) may have passed through a 50 to 500 μm standard sieve.

In one embodiment, the salt concentration of step (b) may be 25 to 27.5 psu.

In one embodiment, the fish of step (c) is flounder ( Paralichthys olivaceus ) , sea bream ( Oplegnathus fasciatus ), red snapper ( Pagrus major ), yellowtail ( Seriola quinqueradiata ), flounder ( Kareius bicoloratus ), flounder ( Pleuronichthys ), flounder ( Pleuronichthys ) Mackerel ( Scomber japonicus ) and anchovy ( Engraulis japonicus ) may be selected from the group consisting of one or more species.

In one embodiment, the exposure time of step (d) may be 50 to 80 hours.

According to the present invention, by controlling the position of the depth of the fish eggs according to the salt concentration of seawater and maintaining a certain interval with the sediments, it is possible to simply evaluate the toxicity by the survival rate and hatching rate of the floating eggs under conditions not affected by microorganisms and bacteria. It has been confirmed that it is possible In addition, unlike the complex analysis method, expensive analysis cost, or the direct use of marine organisms as biochemical indicators, which were disadvantages in the conventional evaluation method of contaminants present in seawater, it is inexpensive and inexpensive using the hatching rate of flounder fertilized eggs. , it has been found that it is possible to easily and conveniently diagnose the contamination of sediments.

Therefore, the method for diagnosing sediment contamination using floating fish eggs of the present invention can be usefully used as an early contamination diagnosis method in the field of marine environmental pollution.

1 is a map and a photograph showing an area from which polluted sediments are collected.
2 is a map and a photograph of an area from which uncontaminated sediments were collected.
3 is a graph showing the hatchability of flounder floating eggs exposed to various salts.
4 is a diagram showing the distance between the sediment and the floating egg in various salinity.
5 is a schematic diagram sequentially illustrating a process of diagnosing contamination of sediment using flounder floating eggs.
6 is a graph showing the hatchability of flounder floating eggs according to the proportion (wt%) of the contaminated sediment in 25 psu and 35 psu seawater.

As used herein, the term "sediment" refers to an insoluble material accumulated on the seabed by moving a rock-origin material separated by weathering and erosion of the rock, a material derived from biological activity, or a chemically formed solid material, among which "Contaminated sediment" is a domestic marine sediment marine environment standard (Ministry of Oceans and Fisheries Notice No. 2016-207. Marine Environment Management Act Article 8), including chemicals that exceed the probable effects level (PEL) for human health or marine ecosystems. "Reference sediments" means seabed sediments that are not polluted below the threshold effects level (PEL) of the domestic seabed sediment marine environment and are inhabited by various organisms. do.

The present invention comprises the steps of: (a) placing a sample containing the sediment into a test chamber; (b) injecting seawater with a controlled salinity concentration into the test chamber, leaving the sediment and seawater in a parallel state, and checking whether the salinity concentration of the seawater in the test chamber is the same; (c) placing the fish eggs into the test chamber; (d) separating the hatched fish individual from the test chamber after the exposure time has elapsed; And (e) provides a method for diagnosing sediment contamination using floating fish eggs, comprising the step of counting surviving individuals from among the separated fish individuals.

In the diagnostic method of the present invention, the step of placing the sample containing the sediment into the test chamber includes a step (ie, step (a)). Step (a) corresponds to S1 shown in FIG. 5 as a step of preparing a marine sediment sample. In the above step, the sediment is the same as described above, and the surface sediment can be collected around the coast using a sampling device such as digger.

The collected sediment can be stored in a plastic container such as polyethylene, frozen, and then thawed before use. The thawed sediment is passed through a standard sieve of 50 to 500 μm, preferably 100 to 400 μm, more preferably 200 to 350 μm, and most preferably 300 μm, and endophytes (e.g., shellfish, worms, etc.) and coarse particles can be removed.

The diagnostic method of the present invention comprises the steps of injecting seawater whose salinity concentration has been adjusted into the test chamber, leaving the sediment and seawater in a parallel state, and checking whether the salinity concentration of the seawater in the test chamber is the same [ that is, step (b)]. Step (b) is a step of injecting seawater with a specific salinity concentration into the test chamber containing the sediment and leaving it for a certain period of time to check whether the salinity of the seawater is not changed by the sediment, which corresponds to S2 shown in FIG. 5 .

The seawater can adjust the salt concentration by using natural seawater or filtered artificial seawater, and it can be prepared by satisfying conditions such as water temperature (15±2 ℃) suitable for hatching of floating flounder eggs, pH conditions, and essential dissolved oxygen. . After adding seawater with a salinity of 25 to 27.5 psu, preferably 25 psu, to the test chamber, it can be left for a certain period of time so that the salinity concentration of the sediment and the seawater become parallel. The salt of 25 psu of the seawater satisfies the survival rate and hatching rate of 80% or more in floating fish eggs, and at the same time, the distance between the sediment and the floating eggs is less than 5 mm, which is the closest to the most suitable conditions for the toxicity test (see FIGS. 3 and 4) ). On the other hand, when the salinity exceeds 27.5 psu, the gap between the sediment and the floating egg is 1 to 6 cm or more, which is not preferable for measuring the degree of contamination of the sediment.

The diagnostic method of the present invention includes the step of placing the floating fish eggs into a test chamber (ie, step (c)). Step (c) corresponds to S3 shown in FIG. 5 as a step of putting the floating fish eggs into a test chamber containing sediment and seawater.

The fish is flounder (Paralichthys olivaceus), rock bream (Oplegnathus fasciatus), red sea bream (Pagrus major), defense (Seriola quinqueradiata), stone flounder (Kareius bicoloratus), flounder (Pleuronichthys cornutus), mackerel (Scomber japonicus) and the anchovies (Engraulis japonicus ) may be selected from the group consisting of, preferably, flounder, which can obtain fertilized eggs from commercial seedling production facilities at low cost throughout the year. After washing the fertilized halibut fertilized eggs (eg, about 0.92±0.05 mm in egg diameter) on the same day of purchase with sterile seawater, a certain number of them may be added per test chamber.

The diagnostic method of the present invention comprises the step of separating the hatched fish individual from the test chamber after the exposure time has elapsed (ie, step (d)). Step (d) corresponds to S4 shown in FIG. 5 as a step of exposing the floating flounder eggs to the sediment during the hatching time of 80% or more.

Halibut floating eggs can be exposed to sediment for 50 to 80 hours, preferably 55 to 70 hours at a water temperature of 15±2 ℃, and most preferably 60 to 70 hours, which is the period during which 80% or more of fertilized eggs of flatfish hatch. The hatching rate can be checked by exposing it to the sediment during the period. If the exposure time is less than 50 hours, the hatching rate of floating eggs is less than 50%, so it is difficult to determine the presence or absence of toxicity. Costs can be wasted.

The diagnostic method of the present invention includes counting the surviving individuals among isolated fish individuals (ie, step (e)). Step (e) is a step of confirming the hatching rate of flounder floating eggs according to the degree of dissolution of the sediment, and corresponds to S5 shown in FIG. 5 .

The hatching rate of the flounder injured eggs can be calculated by counting hatched and non-hatched individuals from the individual separated in step (d), respectively, and then the hatching rate can be calculated based on the surviving individual, and the value obtained by subtracting the number of dead individuals from the total number of hatched individuals is a percentage can be converted to the survival rate.

As shown in FIG. 6 , in seawater with a salinity concentration of 25 psu, a sharp decrease in the hatching rate (34.6%) was observed from 5% by weight of the contaminated sediments, and it was confirmed that the hatching rate was 0% at 10% by weight or more. This decrease in hatching rate is due to the decrease in metabolic activity due to the toxicity of the contaminated sediment, and it is thought that it may affect the growth of the larvae even after hatching. Through this, the method for diagnosing sediment contamination using floating fish eggs of the present invention can be usefully used as a technology for diagnosing contamination of sediments inexpensively, easily and conveniently in the field of marine environmental pollution.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

< Example >

1. Material preparation

(1) Preparation of marine sediment samples

In June 2017, surface sediments were collected from six vertices with a distance of about 1 km along the coast around a nearby factory located in Daejeong-ri, Onsan-eup, Ulju-gun, Ulsan Metropolitan City (see Fig. 1), and at the same vertex several times. The sediments collected over the course of time were mixed to minimize the difference in sediments between the collected samples. Marine sediments with a surface layer of 0 to 2 cm were collected using a scavenger (van-Veen grab sampler), transferred to a polyethylene zipper bag, and stored frozen until testing. After filtering these sediments through a 300 μm standard sieve to remove endogenous organisms and coarse particles, 8 types of heavy metals (arsenic (As)) using domestic marine sediment marine environment standards (Ministry of Oceans and Fisheries Notice No. 2016-207. Marine Environment Management Act Article 8) , cadmium (Cd), chromium (Cr), copper (Cu), mercury (Hg), nickel (Ni), lead (Pb) and zinc (Zn)) were evaluated for contamination.

As shown in Table 1, the sediments were found to exceed the probable effects level (PEL) in all 8 types of heavy metals, and in particular, 6 types (arsenic (As), cadmium (Cd), copper (Cu) ), mercury (Hg), lead (Pb) and zinc (Zn)) were found to be at least 14 times higher than the control standard concentration. As such, highly contaminated sediments were used as samples for this test. According to the above notice, the management standard refers to the concentration with a very high probability of negative ecological effects, and the threshold effects level (PEL) refers to the concentration at which some negative ecological effects are expected to be expressed. In addition, the concentration below the cautionary standard is the concentration predicted to have little negative effect.

Item As CD Cr Cu Hg Ni Pb Zn Management standard (mg/kg) 75.5 2.72 181 64.4 0.62 80.5 119 157 CAUTION Criteria
(mg/kg) 14.5 0.75 116 20.6 0.11 47.2 44.0 68.4 Assay Concentration (mg/kg) 1,100 137 272 4,330 28.1 140 2,960 4,680

Meanwhile, surface sediments from uncontaminated and inhabited areas (Unseo-dong, Jung-gu, Incheon, etc., see Fig. 2) were collected as reference sediments, and for 10 years, the growth, development, propagation and reproduction of marine organisms were affected. It was used in this study, and it was confirmed that all 8 types of heavy metals were below the cautionary standard. The reference sediment was used to dilute the concentration of 0% uncontaminated group and contaminated sediment.

(2) Preparation of test seawater

Uncontaminated natural seawater or artificial seawater filtered with a filter paper of 1 μm or less (containing components of salt similar to natural seawater) was adjusted to a salt concentration of 20, 22.5, 25, 27.5, 30, 32.5 and 35 psu, respectively. of test seawater was prepared. The water quality of the test seawater was measured before the experiment, and it was confirmed that the water temperature was 15±2° C., pH 8.0±0.5, dissolved oxygen 5 mg/L or more, and ammonia 0.2 mg/L or less.

(3) Preparation of flounder floating eggs

Fertilized halibut ( Paralichthys olivaceus ) eggs were sold from seedling producers of various materials such as Bukjeju-gun, Jeonnam and Chungnam in Jeju-si to minimize the difference in sensitivity between seedling production facilities. Floating eggs from which unfertilized eggs, dead eggs, and impurities have been removed from fertilized halibut eggs were washed with sterile seawater and used for the test.

2. Methods and Results

(1) Confirmation of resistance due to salt using floating eggs

A glass chamber (25 ml) for testing was arranged in a constant temperature water bath regardless of the salt concentration, and 20 ml of test seawater of 20, 22.5, 25, 27.5, 30, 32.5 and 35 psu was injected. After placing 10 to 30 flounder eggs per chamber, an increase in salinity in the chamber due to evaporation was prevented by using a stopper. After exposing the prepared flounder eggs to each set salt concentration for 60 hours, the hatching and unhatched individuals were counted, and the hatching rate was evaluated by calculating the percentage for the surviving individuals (95 ~ 100% of the injected floating eggs). This was repeated three or more times and shown in a graph.

As a result of the test, as shown in FIG. 3, the hatching rate of halibut at 25 psu or higher was 80% or higher, and there was no statistically significant difference in the hatching rate according to the salinity of 25, 27.5, 30, 32.5 and 35 psu. ( p > 0.05). Therefore, 25 psu was set as the minimum concentration for which resistance was confirmed in the salinity of the test seawater that satisfies the minimum conditions of 80% or more of survival and hatching rates of controls for toxicity experiments.

(2) Check the distance between the sediment and the floating column

After filling the test chamber with the contaminated sediment to account for about 20% (w/v), 25, 27.5, 30, 32.5, and 35 psu of test seawater are added, and the sediment and the salt of the test seawater are left in equilibrium for a certain period of time. did. Care was taken not to float the sediment when the test seawater was injected into the chamber. After measuring whether the salinity of the test seawater was the same as the set salinity, if it was not the same, the sediment was refilled in a new chamber and the above sequence was repeated. When the salinity of the test seawater was the same as the set salinity, the difference in the distance between the floating flounder eggs and the sediment was evaluated by adding the floating flounder eggs.

In the test seawater of 30 psu or higher, the floating flounder eggs were located at the top of the seawater, at 27.5 psu, at the top of the seawater and in the middle (3 ~ 4 cm) of the sediment, and at 25 psu, it was located at an interval of about 3 to 5 mm from the sediment. (see FIG. 4). In addition, it was confirmed that this phenomenon also appeared in uncontaminated reference sediments.

Therefore, it was confirmed that the salt concentration of 25 psu in seawater is the optimal condition to check the degree of elution of harmful substances according to the distance between the sediment and the floating egg.

(3) Evaluation of contamination of sediment using floating eggs

A method of diagnosing sediment contamination using flounder floating eggs was performed according to the procedure shown in FIG. 5 . According to the proportion (wt%) of polluted sediments using polluted sediments and reference sediments, 0, 0.1, 0.5, 1, 5, 10, 50 and 100 wt% of mixed sediments were prepared as shown in Table 2, and then 2 per ratio Each test chamber was filled to about 20% (w/v).

contaminated sediment
ratio
(weight%) 0 0.1 0.5 One 5 10 50 100 contaminated sediment - 0.1 0.5 One 5 10 50 100 reference sediment 100 99.9 99.5 99 95 90 50 -

The test seawater had a hatching rate of 80% or more, and 25 psu, a salinity concentration that can confirm the elution degree of harmful substances according to the distance between sediment and floating eggs, was used, and compared with 35 psu, which is a general salt concentration of seawater. 25 and 35 psu of test seawater were added, respectively, and the sediment and the salt concentration of the test seawater were allowed to stand for a certain period of time so that the equilibrium state could be reached. After confirming that the salinity of the test seawater was the same as the set salinity, 20 to 50 floating flounder eggs per test chamber were introduced, exposed to salinity concentrations of 25 and 35 psu for 60 hours, and then the floating eggs were separated from the chamber. The hatching and non-hatched individuals were counted from the separated floating eggs, and the hatching rate was evaluated by calculating the percentage for the surviving individuals (95 to 100% of the injected floating eggs), and this was repeated three times and presented in a graph.

As a result of the test, as shown in FIG. 6, the hatching rate decreased sharply from 78.7% to 34.6% as the proportion of contaminated sediment increased from 1% to 5% by weight in the salinity of 25 psu, and at 10, 50, and 100% by weight, the hatching rate was It was confirmed that it was 0%. On the other hand, in the salinity of general seawater, 35 psu, the hatching rate is 80% or more up to 50% by weight of the contaminated sediment, and the hatching rate is 70% or more even at 100% by weight. It was found that the sensitivity of the toxicity assessment was low. Through this, the sediment contamination diagnosis method using the hatching rate of flounder floating eggs at a salinity of 25 psu can be inexpensive, easy and convenient to diagnose whether the sediment is contaminated, and can be usefully used as an early contamination diagnosis method in the field of marine environmental pollution.

Claims (5)

(a) placing a sample comprising the sediment into a test chamber;
(b) injecting seawater with a controlled salinity concentration into the test chamber, leaving the sediment and seawater in a parallel state, and checking whether the salinity concentration of the seawater in the test chamber is the same;
(c) placing the fish eggs into the test chamber;
(d) separating the hatched fish individual from the test chamber after the exposure time has elapsed; and
(e) counting the surviving individuals among the isolated fish populations;
In the method for diagnosing sediment contamination using floating fish eggs comprising The method for diagnosing sediment contamination according to claim 1, wherein the sediment in step (a) has passed through a 50 to 500 μm standard sieve. delete According to claim 1, wherein the fish of step (c) is flounder ( Paralichthys olivaceus ), sea bream ( Oplegnathus fasciatus ), red snapper ( Pagrus major ), yellowtail ( Seriola quinqueradiata ), flounder ( Kareius bicoloratus ), flounder ( Pleuronichthys ) , mackerel ( Scomber japonicus ) and anchovy ( Engraulis japonicus ) A method for diagnosing sediment contamination, characterized in that at least one selected from the group consisting of. The method according to claim 1, characterized in that the exposure time in step (d) is 50 to 80 hours.

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