KR102106698B1 - Method for assessing soil toxicity using nematode offspring counting assay - Google Patents

Abstract

The present invention relates to a method for evaluating soil toxicity using the nematode larval counting method, more specifically, a) exposing an adult nematode to test soil; b) applying the nematode food source to an agar plate; c) placing the test soil containing the nematode of step a) on an agar plate so as not to contact the portion to which the food source is applied; And d) inducing and counting the number of larvae generated from the test soil to the food source.

Description

Method for assessing soil toxicity using nematode offspring counting assay

The present invention relates to a method for evaluating soil toxicity using the nematode larval counting method, more specifically, a) exposing an adult nematode to test soil; b) applying the nematode food source to an agar plate; c) placing the test soil containing the nematode of step a) on an agar plate so as not to contact the portion to which the food source is applied; And d) inducing and counting the number of larvae generated from the test soil to the food source.

Nematodes play an important role in the environmental nutrition system and the nutrition cycle, and are widely distributed in soil and microbial-rich habitats. The independent nematode, Caenorhabditis elegans , is a standardized, short-lived developmental stage and the only animal with a fully known cellular and nervous system. Therefore, the pretty little nematode is generally known as a key species for use in toxicity testing.

Williams and Dusenbery (1990), Aquatic toxicity testing using the nematode, Caenorhabditis elegans ] (Environ. Toxicol. Chem. 9, 1285-1290.), a variety of studies have been using these bioassays since the first water test method using nematode nematodes was first presented. The nematode toxicity test method was also performed in soil media, and various studies have reported the development of this type of bioassay using whole sediment and soil samples obtained from natural sites.

Since the American Society for Testing and Materials (ASTM) and the International Organization for Standardization (ISO) established standardized nematode toxicity testing methods (ASTM, 2001; ISO, 2010), studies have routinely used these methods. Recently, nematode nematodes have been widely used to evaluate the toxicity mechanisms of various biocomposites, including environmental hormones and metal-based nanomaterials, and bioassays have also been used to evaluate complex exposure scenarios such as multi-generational or nutrient delivery effects. come.

In these prior studies, agar and liquid were selected as test media, but no soil substrate was used. In a standard toxicity test using nematodes, pretty nematode individuals are exposed to contaminated soil and sediment systems and extracted from the medium by a colloidal silica flotation method using LUDOX solution (ASTM, 2001; ISO, 2010). The ISO (2010) standard method includes a Rose Bengal dyeing method to improve recovery.

Although these methods have been internationally standardized, there are limitations in reporting toxicity values. Prior studies using ISO standards have focused on interpreting the relationship between media properties and toxicity, application methods for natural samples, and standardization methods.

Studies using ASTM standards were designed to evaluate the lethal or arch death effects of various metal toxic substances on nematodes in soil media (ASTM, 2001).

To determine the level of protection against ecological receptors in soil environments, a stochastic ecological risk assessment (PERA) is performed using species sensitivity distribution (SSD) to evaluate and quantify data (ECB, 2003; NEPC, 2011). . For this step, the use of various ecological toxicity data from bioassay batteries is very important, and the calculations should be performed carefully and reliably using standardized methods. Although ISO and ASTM standard methods have already been published and provide reliable data, there is a growing need for derivation of toxicity values, rapid methods and accessibility of researchers.

On the other hand, Republic of Korea Patent Publication No. 10-2012-0041063 using a functional genome technology, especially RNAi technology of the small nematodes, biomarkers for toxic substances through inductive analysis of the correlation between the sensitive indicator and the physiological level of the individual It has been disclosed a method for screening and searching for environmental toxic substances using the same.

However, no method has been proposed for evaluating soil ecological toxicity by nematode larval counting.

Thus, the present inventors exposed the nematodes to the soil using a standardized toxicity test method, and then counted the larvae formed in the test soil attracted by the food source, as well as being faster and simpler than the conventional soil ecological toxicity evaluation method. , The present invention was completed by confirming that a more sensitive toxicity value can be induced.

Accordingly, an object of the present invention is to provide a method for evaluating soil toxicity using nematode larval counting.

In order to solve the above-described problems, the present invention provides a soil toxicity evaluation method comprising the following steps: a) exposing adult nematodes to test soil; b) applying the nematode food source to an agar plate; c) placing the test soil containing the nematode of step a) on an agar plate so as not to contact the portion to which the food source is applied; And d) deriving and counting the number of larvae from the test soil into the food source.

According to one preferred embodiment of the present invention, the nematode of step a) may be a pretty little nematode ( Caenorhabditis elegans ).

According to another preferred embodiment of the present invention, the nematode in the adult state of step a) may be an adult-synchronized adult for 54 to 58 hours in an egg state.

According to another preferred embodiment of the present invention, the test soil of step b) may be arranged linearly in the center of an agar plate.

According to another preferred embodiment of the present invention, the microbial food source of step c) may be E. coli.

According to another preferred embodiment of the present invention, the coefficient of step d) may be performed 1 to 3 hours after performing step c).

According to another preferred embodiment of the present invention, the method can be carried out under ventilated conditions.

According to another preferred embodiment of the present invention, the soil toxicity may be toxicity to the metal.

According to another preferred embodiment of the present invention, the metal may be at least one selected from the group consisting of arsenic, cadmium, copper, nickel, lead and zinc.

The method for evaluating soil ecological toxicity using the nematode larval counting method of the present invention can be used as a quick and simple bioassay than a conventional method. The method of the present invention can be performed within 60-90 seconds per replicate and has the obvious advantage of keeping the operator's hands free for 3 hours before larval measurement. The nematode larva counting method of the present invention also includes minimal additional stress on living species and enables secondary toxicity tests such as oxidative stress, genetic analysis, and multi-generational analysis, making it more sensitive to toxicity compared to conventional methods. Make it possible to derive the value.

Figure 1 shows a schematic diagram of the nematode larval counting method using the pretty little nematode of the present invention.
Figure 2A is a schematic representation of a soil-agar separation plate designed for use in the nematode larval counting method of the present invention, Figure 2B is a schematic representation of a larva recovered from a soil-agar separation plate, and Figure 2C is a soil -It shows the process of placing the test soil using agar separation plate.
FIG. 3A shows the time-dependent change in larval number on soil-agar separation plates with or without food sources, and FIG. 3B shows the number of larvae in LUFA 2.2 in 50 replicate experiments. The solid white line and the orange area indicate the mean value and the standard deviation, respectively. The average, maximum, and minimum values of the larvae were calculated as 124 ± 36, 192, and 42, respectively.
Figure 4 shows the change in the number of larvae of pretty little nematodes exposed to various metals in LUFA 2.2 soil, A is arsenic (As), B is cadmium (Cd), C is copper (Cu), D is nickel (Ni) , E represents lead (Pb), and F represents zinc. Data were expressed as a percentage of the control group, and a significant difference (*) compared to the control group was calculated using the Dunnet program ( p <0.05). Error bars indicate standard deviation, and EC50 values of each metal were calculated by the trimmed Spearman-Karber method.
FIG. 5 shows the number of larvae generated from 36 field soil samples (S1-S36), and solid white lines and orange areas indicate mean values and standard deviations, respectively. The mean, maximum, and minimum values of the larvae were calculated to be 41 ± 26, 98 (S35), and 1 (S5), respectively.

Hereinafter, the present invention will be described in more detail.

As mentioned above, although ISO and ASTM standard methods have already been published and provide reliable data, there is a growing need for derivation of toxicity values, rapid methods and accessibility of researchers. Thus, the present inventors exposed the nematodes to the soil using a standardized toxicity test method, and then supplied a food source to count larvae formed in the test soil attracted by the food source, which is faster than the conventional soil ecological toxicity evaluation method. The present invention was completed by confirming that it was not only simple and simple, but also capable of inducing more sensitive toxicity values.

The method for evaluating soil ecological toxicity using the nematode larval counting method of the present invention can be used as a quick and simple bioassay than a conventional method.

Accordingly, the present invention provides a method for evaluating soil toxicity comprising the following steps:

a) exposing adult nematodes to test soil;

b) applying the nematode food source to an agar plate;

c) placing the test soil containing the nematode of step a) on an agar plate so as not to contact the portion to which the food source is applied; And

d) counting by deriving the number of larvae from the test soil to the food source.

In the method for evaluating soil toxicity of the present invention, it is preferable that the nematode of step a) is a nematode nematode ( Caenorhabditis elegans ), and the nematode nematode is preferably an adult tuned for 54 to 58 hours in an egg state.

The soil toxicity evaluation method of the present invention is based on a method using a soil-agar separation plate, for example, the soil-agar separation plate may use an NGM agar plate, but is not limited thereto.

The optimum conditions for the soil-agar separation method are linearly placed soil, the presence of food and ventilation, and it is preferable that the test soil is specifically placed linearly in the center of the agar plate.

In the method for evaluating soil toxicity of the present invention, the microbial food source in step c) may be E. coli, for example, E. coli strain OP50 may be used, but is not limited thereto.

In the soil toxicity evaluation method of the present invention, since the number of larvae generated from the soil was found to be stable in 1 to 3 hours, the coefficient of step d) was performed after 1 to 3 hours after performing step c), Preferably it can be carried out after 3 hours.

The method for evaluating soil toxicity of the present invention is a highly efficient nonchemical method for isolating live nematodes ( Caenorhabditis ) in Kim et al. (2014) in that its endpoint is "number of offspring". elegans ) from soil during toxicity assays] (Environ. Toxicol. Chem. 34, 208-213) (hereinafter referred to as 'Kim et al. (2014) literature') is different from the conventional method of counting the number of live adults. .

In addition, the method used in the literature of Kim et al. (2014) periodically recovers the adult for 24 hours after placing the test soil containing nematodes on the soil-agar separation plate, but the soil toxicity evaluation method of the present invention uses the test soil. It has a very advantageous effect in that the toxicity of the soil can be evaluated by counting the number of larvae only once after 1 to 3 hours after placement, preferably after 3 hours.

Soil toxicity evaluation method of the present invention is to evaluate the chemical toxicity of the soil, preferably metal soil toxicity, the metal may be at least one selected from the group consisting of arsenic, cadmium, copper, nickel, lead and zinc, , But is not limited to this.

The applicability of the method for evaluating soil toxicity using the nematode larva counting method of the present invention was verified by using metal mixed soil as described in Example 2, and the number of counted larvae decreased depending on the metal concentration as shown in FIG. 4. Was confirmed.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

eelworm  Larval counting

1.1 Test  Biology

The pretty little nematode (wild type, Bristol line N2) was obtained from Konkuk University Animal Genome Laboratory (Seoul, Korea). All nematodes are source of nematode growth media containing E. coli strain OP50 (NGM: NaCl 3 g / L, peptone 2.5 g / L, agar 17 g / L, 1 M potassium phosphate 25 mL / L, 1 M CaCl ₂ 2H ₂ O 1 mL / L, 1 M MgSO ₄ · 7H ₂ O 1 mL / L, cholesterol 1 mL / L) (Brenner, 1974). The E. coli strain was obtained from the Korea Agricultural Microbiological Resource Center (KACC, Seoul, Korea). Incubation was maintained at 20 ± 2 (SE) ° C under no light conditions.

To obtain an age-synchronized nematode, at least 3 days old nematodes were treated with a chlorox solution for 10 minutes, and the solution was centrifuged at 400 × g for 2 minutes. The treated samples were washed three times with K-medium (0.032 M KCl, 0.051 M NaCl) (Williams and Dusenbery, 1990) and pellets of eggs were applied to NGM medium containing E. coli OP50. Plates were incubated in the incubator until the initial adult stage (54-58 hour synchronisation after treatment with Clorox solution) under conditions of no light at 20 ° C. All components of the medium except sodium chloride and potassium chloride (Deoksan, Asan, Korea) were purchased from BD (Maryland, USA).

1.2 eelworm  Larval counting

LUFA (Landwirtschaftliche Untersuchungs-und Forschungsanstalt) 2.2 (LUFA Speyer, Germany) was chosen as the test soil for evaluating the usefulness of nematode larval counting. According to the supplier's instructions, the pH, organic matter (OM) content, organic carbon content and texture type were 5.5 ± 0.2, 3.82%, 1.77%, and roaming sand, respectively. The LUFA 2.2 soil was sieved through a 2 mm sieve and air dried for 7 days. K-medium (0.2 mL) was added to each well of a 24-well plate containing 0.3 g of LUFA 2.2 soil, and 10 synchronized adults were added. After 24 hours, soil containing nematodes was spread on a soil-agar separation plate (Kim et al. (2014)).

To prepare a soil-agar separation plate, E. coli strain OP50 was cultured in LB medium (Luria-Bertani medium, 25 g / L) at 37 ° C for 16 hours, and 150 μL of cell suspension (as shown in FIG. 2A). Optical density 1.1-1.2, 8.0-9.6 Х 10 ⁸ cells / ml) were plated on NGM agar medium. As shown in Figure 2B, the test soil was placed linearly in the central region of the soil-agar separation plate, and the larvae from the soil were counted. The placement of the test soil should be performed with great care and accuracy, and the contact between the feeding area and the soil boundary should be minimized.

As shown in Fig. 2C, first, an experimental spatula is used to transfer a lump of soil that can be excavated to a soil-agar separation plate, and the remaining water containing soil particles and nematodes is tensioned between the well plate and the experimental spatula. It was moved using. 50 μL of K-medium was added to prevent water loss. After 0.5, 1, 2, 3, 4, and 5 hours, larvae from the test soil were counted microscopically (n = 3). To assess whether the absence of food affects larval recovery, soil-agar separation plates that do not contain food sources were also prepared ( n = 4). The optimum observation point (h) was determined, and the average value of the larva was calculated based on 50 repeated experiments.

After larval development from LUFA 2.2 soil, nematode larval counting was performed, and they showed very active behavior in the microbial food zone.

As shown in Figure 3A, the number of larvae on the soil-agar separation plates containing food increased time-dependently (148 ± 7, 177 ± 23, respectively at 0.5, 1, 2, 3, 4, and 5 hours). 177 ± 29, 177 ± 22, 181 ± 15, and 199 ± 38 nematodes). In contrast, the number of larvae from soil-agar separating plates lacking food decreased time-dependently (90 ± 23, 88 ± 8, 78 ± 9, respectively at 0.5, 1, 2, 3, 4, and 5 hours). 66 ± 7, 54 ± 5, and 21 ± 4 nematodes).

Since the number of larvae that appeared occurred to be stable at 1 to 3 hours, the optimal observation time was determined to be 3 hours. To determine the mean value of the larva for LUFA 2.2, the test was repeated 50 times. As a result, as shown in FIG. 3B, the average value was calculated as 124 ± 36 nematodes, and the maximum and minimum values were determined as 192 and 42 nematodes, respectively.

In the present invention, the nematode larval counting method was performed by slightly modifying the method described in the previous study (Kim et al. (2014)). This conventional study suggested a soil-agar separation method for recovering live nematodes. In accordance with standard test guidelines (ASTM, 2001; ISO, 2010), test soil containing pretty little nematodes used for exposure was placed on an NGM agar plate, and a microbial food source was used to attract live nematodes from the soil. .

Using the method of the present invention, it has been found that the larval counting method is more efficient and faster than the live adult counting method of the previous study, and detailed methods have been investigated for test standardization.

Optimal conditions for the soil-agar separation method are reported in the literature of Kim et al. (2014): linearly placed soil, the presence of prey, and ventilation.

As previously identified, the number of larvae generated in the soil-agar separation plate containing food increased in a time-dependent manner, while the number of larvae generated in the soil-agar separation plate without food decreased in a time-dependent manner (FIG. 3A). The difference between the presence and absence of food may be related to the chemotaxis of the pretty little nematode to the smell of food, and accordingly, the inventors have concluded that the food source of the soil-agar separation plate is an essential condition for nematode larval counting.

The selection of a representative control group is very important in the toxicity assessment because it requires comparable data for reliable toxicity quantification. In the present invention, LUFA 2.2 soil widely used in soil ecological toxicity studies was selected as a control soil (Løkke and Van Gestel, 1998).

As indicated previously, the average value of larvae from uncontaminated LUFA 2.2 was determined to be 124 ± 36 nematodes (Figure 3B), which corresponds to the number of larvae of 12.4 ± 3.6 per adult. The inventors used 54 to 58 hours synchronized nematodes for 24 hour toxic bioassays, thus obtaining larvae from adult grown 78 to 82 hours. The nematode nematode N2 strain generally lays eggs at an average generation of 73.0 hours (68.5-80.5 hours), and reproductive activity lasts up to 118 hours at 20 ± 1 ° C. Muschiol et al. (2009), Life cycle and population growth rate of Caenorhabditis. elegans studied by a new method.] (BMC Ecology 9, 14.) reported that an 80.5-hour generation of nematodes can produce 17.8 eggs, which hatch within 7.3 hours (5.0-10.6 hours). . Given that the hatching rate is about 79.6 ± 7.1% at 20 ° C, this information is consistent with the results for the average number of larvae per adult (12.4 ± 3.6).

The ISO standard method shows that the effectiveness criterion for the regulation of reproductive activity is 30 larvae and 80% fertility per nematode, which are representative values during a 96-hour trial period using stage 1 childhood nematodes (ISO, 2010). According to conventional studies on larva production in soil and sediment media, the number of larvae showed high variability in the range of 44 to 144 larvae per nematode in various reference soils (Hφss et al. (2009)) , The number of larvae produced per nematode in the sediments and soil of the control group was reported to be 56 to 235 and 54 to 165, respectively (Hφss, etc.) (2012)). Since these studies performed nematode bioassays using ISO standard procedures, the number of larvae obtained cannot be directly compared with the results of the present invention.

eelworm  Applicability of the larval counting method

To evaluate the applicability of nematode larval counting for toxicity evaluation, we investigated its usefulness by testing soils modified with metal. Arsenic (As), cadmium (Cd), copper (Cu), nickel (Ni), lead (Pb), and zinc (Zn) soils at LUFA 2.2 0, 200, 400, 600, 800, and 1,000 mg / kg It was prepared at a concentration of. Test chemical, _{NaAsO 2, CdCl 2, CuCl 2} · 2H 2 O, NiCl 2, PbCl 2, and ZnCl ₂ were obtained from Sigma (Sigma), a stock solution was prepared from each culture medium K- metal in the soil (As , Cd, Cu, Ni, Pb, and Zn).

Soil samples (0.3 g) were placed in each well of a 24-well plate ( n = 3) and 0.2 mL of a stock solution containing metal. The final concentration of soil was determined for each of the aforementioned concentrations (0, 200, 400, 600, 800, and 1,000 mg / kg). Ten nematodes were added to each well and exposed for 24 hours at 20 ° C without light. The present inventors used the nematode counting method at the end of the test, and counted the number of larvae generated from the test soil after 3 hours.

As a result, as shown in Table 1, the number of caterpillars occurring in all the control groups was 104 to 157, and it was decreased depending on the metal concentration.

The number of larvae of nematode nematodes exposed to As, Cd, Cu, Ni, Pb, and Zn density
(mg / kg) Larvae As CD Cu Ni Pb Zn 0 104 ± 24 119 ± 24 157 ± 17 143 ± 8 134 ± 19 115 ± 25 200 78 ± 6 126 ± 18 150 ± 37 145 ± 10 103 ± 46 86 ± 29 400 34 ± 25 103 ± 8 94 ± 28 116 ± 15 119 ± 40 84 ± 50 600 14 ± 2 71 ± 10 33 ± 6 101 ± 12 89 ± 35 76 ± 30 800 9 ± 5 29 ± 15 18 ± 7 59 ± 9 94 ± 43 51 ± 15 1000 3 ± 3 25 ± 9 5 ± 3 44 ± 8 71 ± 23 34 ± 19

The data for the contaminated soil is shown in FIG. 4 as a percentage of the number of larvae generated relative to the number of control larvae that occurred. In the group exposed to As, the amount of larvae that occurred was 75 ± 5%, 33 ± 24%, 14 ± 2%, 8 ± 4% in soils containing 200, 400, 600, 800, and 1,000 As mg / kg, respectively. , And 3 ± 2% (FIG. 4A). Even when exposed to other metals in the same range of concentration, larval development decreased concentration-dependently: 106 ± 15%, 87 ± 7%, 59 ± 9%, 25 ± 12%, and 21 in the group exposed to Cd. ± 7% (Figure 4B); 96 ± 24%, 60 ± 18%, 21 ± 4%, 11 ± 5%, and 3 ± 2% in the group exposed to Cu (FIG. 4C); 101 ± 7%, 81 ± 10%, 70 ± 9%, 41 ± 6%, and 31 ± 5% in the group exposed to Ni (FIG. 4D); 76 ± 26%, 89 ± 21%, 66 ± 25%, 70 ± 27%, and 53 ± 9% in the group exposed to Pb (FIG. 4E); And 73 ± 27%, 68 ± 36%, 62 ± 20%, 43 ± 10%, and 28 ± 15% in the group exposed to Zn (FIG. 4F).

As shown in Figure 4, the 50% effective concentration (EC ₅₀ ) value for the number of larvae that occurred was 303 (272-338) As mg / kg, 637 (593-684) Cd mg / kg, 429 (400-460) It was calculated as Cu mg / kg, 744 (688-804) Ni mg / kg,> 1,000 Pb mg / kg, and 669 (572-782) Zn mg / kg. As a result of using the nematode larva counting method of the present invention, the most toxic metal in LUFA 2.2 soil was determined as As, followed by Cu, Cd, Zn, Ni, and Pb.

As described above, the present inventors performed the nematode larval counting method to evaluate the chemical toxicity of the metal-modified LUFA 2.2 soil. When using the method of the present invention, the number of caterpillars occurred varied, and the toxicity tendency for each metal could be classified.

Nematode toxicity tests using soil and sediment media were standardized by ASTM and ISO (ASTM, 2001; ISO, 2010), but determination of toxicity values such as EC50 and 50% lethal concentration (LC50) was much more difficult than expected. ISO standard methods have been developed based on a variety of studies using whole sediment and soil samples from natural sites, and recently some studies have reported the assessment of pyrene and Zn toxicity using this method. However, these studies focused on interpreting the relationship between media properties and toxicity, how natural samples were applied, and how to standardize the method. Another standard test method, the ASTM standard method, was designed to assess the lethal or arch death toxic effects on nematodes in soil media (ASTM, 2001). Document of a pretty little for the first time Donkin Dusenbery and soil toxicity test method using a nematode (1993) [A soil nematode Caenorhabditis elegans and the toxicity test using an effective method of recovery] (Arch. Environ. Contam. Toxicol. 25, 145- 151.), and this method was routinely used until 2009.

Most studies performed nematode bioassays using soils with contaminants and predicted their toxicity in terms of toxicity values (eg, LC50). Donkin and Dusenbery (1993) used Cu as the sample material, and they reported LC50 ranging from 413 to 1061 mg / kg for various natural soils.

Peredney and Williams (2000b), Utility of Caenorhabditis elegans for assessing heavy metal contamination in artificial soil] (Arch. Environ. Contam. Toxicol. 39, 113-118.) was 1,641 for Cd, Cu, Ni, Pb, and Zn, respectively, during 24 hours of exposure in ASTM soil. , LC50 values of 1,272, 2,490, 2,293, and 1,915 mg / kg were reported. These toxicity values cannot be directly compared with the results of the present invention, but through this, the inventors can conclude that the nematode larval counting method of the present invention can derive more sensitive toxicity values using a faster and simpler process.

Field soil used eelworm  Application of larval counting method

The inventors investigated the applicability of nematode larval counting methods using 36 field soil samples (S1-S36).

36 field soils (S1-S36) were collected from field sites containing 3 metals in Korea. These sites are located around the arsenic smelter, and have been contaminated with dust from the smelter chimney. High levels of arsenic (As), cadmium (Cd), and lead (Pb) were detected in soil in a nearby area (2 km) centered around the chimney.

The soil was separated by hand, air dried and sieved through a sieve of <2 mm. The soil stability (AS) and the bulk density (BD) of Friedman (2001) et al. (Guidelines for Soil Quality Assessment in Conservation Planning) (Joubert, B., Ed., NRCS SQI, Washington, DC, USA) And Tan (2005), Measurement of bulk density. In Soil Sampling, Preparation, And Analysis] (2nd ed. Dekker M, CRC Press, New York, pp 178.). The cation exchange capacity (CEC) was evaluated by extraction using ammonium acetate and potassium chloride, and the electrical conductivity (EC) and pH were respectively EC meter (Multi Meter CP-500L; Istech, Seoul, Korea) and heat loss (1: 5 Extraction). Soil texture (percentage of clay, sand and silt) and water volume (WHC) were tested using the soil texture test kit (1067 model; Lamot, Chestertown, Maryland, USA) and standard methods (Harding and Ross, 1964; USDA, 2001). It was evaluated using.

The experimental results are shown in Table 2 below.

Physicochemical properties of 36 (S1-S36) field soils characteristic Stability Bulk density Cation
exchange
Volume Electric
conductivity Organic matter pH sand Silt clay Water Abbreviation AS BD CEC EC OM pH Sand Silt Clay WHC unit % g / ml Cmol ⁺ / kg dS / m % unit
none % % % ml / g S1 84.0 1.4 2.0 1.2 0.7 7.5 88.0 0.0 12.0 0.5 S2 85.0 1.2 9.0 6.3 7.4 5.8 8.0 58.0 34.0 0.7 S3 72.0 1.5 3.0 5.1 2.7 8.3 86.0 0.0 13.0 0.4 S4 73.0 1.3 3.0 0.5 1.6 7.4 77.0 7.0 16.0 0.5 S5 53.0 1.5 3.0 6.3 1.5 4.2 74.0 9.0 17.0 0.4 S6 49.0 1.0 5.0 1.1 4.3 6.3 7.0 56.0 37.0 0.7 S7 58.0 1.1 10.0 3.7 2.2 5.9 4.0 63.0 33.0 0.7 S8 79.0 1.4 4.0 1.0 1.2 8.3 87.0 0.0 13.0 0.4 S9 54.0 1.5 3.0 6.0 1.3 8.9 77.0 16.0 7.0 0.3 S10 68.0 1.1 20.0 1.9 3.5 7.9 17.0 28.0 55.0 0.7 S11 63.0 1.2 17.0 10.2 2.7 10.2 8.0 39.0 53.0 0.8 S12 61.0 1.3 17.0 3.0 5.0 9.4 13.0 33.0 54.0 0.5 S13 63.0 1.2 23.0 1.1 2.6 7.1 5.0 29.0 66.0 0.9 S14 58.0 1.2 24.0 2.2 3.3 6.7 5.0 44.0 51.0 0.9 S15 63.0 1.3 18.0 4.6 3.7 10.0 8.0 38.0 54.0 0.6 S16 44.0 1.4 16.0 0.8 2.5 7.6 44.0 35.0 21.0 0.5 S17 61.0 1.3 17.0 7.4 3.6 7.5 74.0 1.0 25.0 0.5 S18 47.0 1.3 19.0 7.8 7.8 6.7 57.0 1.0 42.0 0.5 S19 27.0 1.4 17.0 0.5 3.0 7.3 37.0 37.0 26.0 0.6 S20 52.0 1.4 17.0 1.0 6.9 7.2 39.0 6.0 55.0 0.5 S21 23.0 1.4 16.0 1.8 2.1 6.1 48.0 25.0 27.0 0.5 S22 60.0 1.2 23.0 2.8 5.2 6.3 10.0 15.0 75.0 0.7 S23 60.0 1.3 24.0 3.1 6.3 6.3 12.0 11.0 77.0 0.7 S24 53.0 1.4 16.0 0.5 6.0 5.3 10.0 59.0 31.0 0.5 S25 57.0 1.2 8.0 0.9 5.8 5.4 61.6 38.1 0.3 0.5 S26 54.0 1.4 4.0 1.9 2.2 7.5 78.8 20.9 0.3 0.4 S27 63.0 1.2 7.0 20.8 5.5 5.4 64.2 35.3 0.5 0.5 S28 76.5 1.4 10.0 0.4 1.9 5.6 48.1 18.6 33.3 0.5 S29 79.3 1.3 15.0 3.2 2.1 6.8 42.5 15.5 41.9 0.6 S30 77.4 1.1 14.7 1.3 5.5 6.5 51.3 15.6 33.1 0.6 S31 68.2 1.5 6.8 25.3 2.3 7.1 61.1 10.1 28.8 0.4 S32 65.3 1.5 6.7 0.8 1.5 9.6 61.9 4.6 33.5 0.4 S33 58.1 1.6 6.8 8.4 1.1 5.7 77.7 8.1 14.2 0.4 S34 57.0 1.6 6.8 0.2 0.9 7.1 86.6 3.3 10.1 0.3 S35 65.6 1.3 10.3 12.7 4.2 5.5 82.9 7.1 10.1 0.5 S36 65.7 1.3 10.1 0.7 3.7 6.5 82.3 6.8 10.9 0.5

The total metal concentration (As, Cd, Cu, Ni, Pb, and Zn) of each soil sample was determined using an acid decomposition method, and then inductively coupled plasma atomic emission spectrophotometers (ICP-AES; JY 138, Ultrace, Jobin) Yvonne, France), the compounds were analyzed, and the results are shown in Table 3 below.

Metal concentrations of As, Cd, Cu, Ni, Pb, and Zn for 36 (S1-S36) field soils characteristic arsenic cadmium Copper nickel lead zinc Abbreviation As CD Cu Ni Pb Zn unit mg / kg mg / kg mg / kg mg / kg mg / kg mg / kg S1 15.6 0.7 42.2 20.5 57.6 88.2 S2 20.7 0.3 33.6 21.4 42.4 85.1 S3 21.0 0.6 66.3 37.4 97.7 144.4 S4 18.6 ND 31.5 9.6 52.5 48.7 S5 34.0 ND 58.4 14.7 111.9 90.3 S6 31.2 ND 44.1 21.8 48.5 89.7 S7 10.1 ND 18.7 17.7 32.8 69.0 S8 24.3 ND 97.4 10.4 125.0 340.2 S9 16.4 ND 69.9 12.8 76.6 130.6 S10 12.9 ND 40.2 17.5 59.4 90.1 S11 19.6 ND 22.2 14.4 34.8 121.3 S12 18.3 ND 17.5 14.2 29.8 80.7 S13 20.8 ND 14.9 16.7 27.6 66.6 S14 26.9 ND 19.1 17.6 32.2 69.1 S15 17.2 ND 14.3 13.6 29.0 65.1 S16 12.4 ND 22.3 26.0 31.4 83.4 S17 8.0 ND 71.9 10.6 41.5 112.9 S18 17.5 ND 44.7 14.2 60.4 141.0 S19 23.4 ND 57.9 17.6 88.5 98.5 S20 17.5 ND 43.3 17.9 68.7 88.7 S21 5.8 ND 10.9 16.6 27.9 73.7 S22 38.0 ND 26.7 18.1 51.6 77.6 S23 30.0 ND 29.7 19.5 51.1 82.2 S24 6.3 ND 27.5 88.6 30.6 82.3 S25 502.5 ND 113.3 32.6 98.2 110.3 S26 43.5 ND 45.3 9.5 96.3 124.0 S27 22.5 ND 53.7 112.5 35.9 87.5 S28 35.3 ND 59.8 41.5 17.8 85.7 S29 24.6 ND 60.2 65.3 36.3 86.1 S30 19.1 ND 44.4 50.6 27.6 67.1 S31 35.4 ND 28.6 24.6 53.2 90.7 S32 49.4 ND 31.8 22.6 52.6 94.6 S33 8.0 ND 16.1 18.7 25.7 58.0 S34 5.5 ND 14.1 19.1 21.9 65.2 S35 6.7 ND 12.2 17.3 20.3 52.2 S36 15.3 ND 12.9 12.4 24.9 56.4

(ND = not detected)

Each metal has a standard curve of 3-4 points by ICP-AES analysis, and each R ² value is 0.99993, 0.99993, 0.99996, 0.99997, 0.99997, respectively by As, Cd, Cu, Ni, Pb, and Zn analysis, respectively. And 0.99995. The detection limits of As, Cd, Cu, Ni, Pb, and Zn using ICP-AES were 0.5, 0.05, 0.05, 0.05, 0.1, and 0.05 μg / L, respectively. To apply nematode larva counting to field soil, 0.3 g of air dried soil sample was added to wells of a 24-well plate and moistened. To control the moisture content, the WHC of each soil sample was measured and the moisture content was adjusted to 142% WHC (the same moisture content as in the LUFA test) for each sample. The hydrated soil of the 24-well plate was placed at 20 ° C without light for 24 hours for equilibration. After equilibration, 10 synchronized nematodes were exposed to the prepared soil for 24 hours, and nematode larval counting was performed as mentioned above.

As a result, as shown in FIG. 5, the number of larvae originating from the field soil of S1 to S36 was determined as follows: 44 ± 12, 14 ± 4, 37 ± 15, 58 ± 17, 1 ± 2, 49 ±, respectively. 18, 18 ± 9, 57 ± 7, 4 ± 6, 17 ± 9, 58 ± 19, 50 ± 16, 10 ± 5, 15 ± 15, 34 ± 13, 30 ± 2, 25 ± 15, 64 ± 24, 23 ± 12, 44 ± 11, 14 ± 4, 48 ± 10, 28 ± 10, 15 ± 5, 14 ± 3, 96 ± 11, 46 ± 5, 86 ± 5, 47 ± 6, 70 ± 16, 52 ± 30, 41 ± 12, 87 ± 15, 28 ± 15, 98 ± 12, and 57 ± 14 animals. The mean, maximum and minimum values for the number of larvae from all field samples were determined to be 41 ± 26, 98 ± 12 (S35), and 1 ± 2 (S5), respectively.

Some data could not be explained by metal contamination and its toxicity in nematode nematodes. For example, field soils S2, S7, S13, and S24 had fewer larvae (14 ± 4, 18 ± 9, 10 ± 5, and 15 ± 5 nematodes, respectively), which were not related to metal concentration, These field soils exhibited metal concentrations lower than the average of the total data set (As 34.3 mg / kg, Cd 0.5 mg / kg, Cu 39.4 mg / kg, Ni 25.4 mg / kg, Pb 50.6 mg / kg, and Zn is 94.4 mg / kg) (Table 3). Soil S8 also showed a low correlation between Zn concentration (340.2 mg / kg) and larval data (57 ± 7 nematodes).

Soil is highly volatile because it is a complex medium with a wide range of interactions between different properties. The results for the field soils S2, S7, S8, S13, and S24 are interpreted to appear due to such soil variability, but for the remaining field soils, soil toxicity can be evaluated with high tendency using the nematode larval counting method of the present invention. Was confirmed. Therefore, it can be seen that the nematode larval counting method of the present invention is a useful tool for evaluating soil toxicity by applying to real field soil, and can evaluate toxicity with a higher sensitivity than conventional soil toxicity evaluation methods.

Data analysis

The Dunnett's Program ver. 1.5 was used to calculate the significant difference ( p <0.05) between the NOEC (no-observed-effect concentration) and the mean value of the control and treatment groups. Median effect concentration (EC ₅₀ ) values were calculated by the Trimmed Spearman-Carver method.

Claims (9)

a) exposing the adult nematode to the test soil;
b) applying the nematode food source to an agar plate;
c) placing the test soil containing the nematode of step a) on an agar plate so as not to contact the portion to which the food source is applied; And
d) counting the number of offspring generated from the test soil 1 to 3 hours after being induced to the food source;
As a soil toxicity evaluation method comprising,
The end point (endpoint) of the soil toxicity evaluation method is the number of larvae, soil toxicity evaluation method. The method for evaluating soil toxicity according to claim 1, wherein the nematode of step a) is a pretty nematode ( Caenorhabditis elegans ). The method for evaluating soil toxicity according to claim 1, wherein the nematode in the adult state of step a) is an age-synchronized adult in an egg state for 54 to 58 hours. The method for evaluating soil toxicity according to claim 1, wherein the test soil in step c) is arranged linearly in the center of an agar plate. The method of claim 1, wherein the food source of step b) is E. coli. delete The method for evaluating soil toxicity according to claim 1, wherein the method is performed under ventilation conditions. The method for evaluating soil toxicity according to claim 1, wherein the soil toxicity is toxic to metal. The method for evaluating soil toxicity according to claim 8, wherein the metal is at least one selected from the group consisting of arsenic, cadmium, copper, nickel, lead, and zinc.

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