Developing a Safety Management Method for Endosulfan Using Biochar in Ginseng Fields

Abstract

Endosulfan is an endocrine disruptor that negatively affects the human central nervous system. Although perennial root vegetable crops have high risks of endosulfan absorption and transfer in soil, safety management studies addressing this problem are lacking. We evaluated endosulfan absorption and transition, as well as plant growth in ginseng cultivation soil, and developed a safety management method for field application. Total endosulfan residual concentrations in the soil and biochar 0.1–1.0% treatment groups were 52–73% after 532 d of spraying, and there was no reduction effect owing to biochar treatment. However, the endosulfan sulfate conversion rate decreased by 21.6–47.1% as the biochar amount increased. Further, there was a 47–95% reduction in the absorption and migration of endosulfan into ginseng in the biochar treatment compared to the control, demonstrating a reduction effect (p < 0.05). Ginseng grown in soil treated with 0.1% biochar showed no growth parameter differences compared to the control (p > 0.05); however, germination rates decreased to <59% when the soil was treated with ≥0.3% biochar. Soil treatment with 0.1% biochar can reduce endosulfan absorption and migration without adversely affecting crop growth. This treatment can be used at the cultivation site, depending on soil conditions.

1. Introduction

For agricultural products, substances for which the maximum residue limit has not been set have been collectively applied at 0.01 mg kg⁻¹ since the implementation of the positive list system and zero tolerance system [1,2]. As a result, highly persistent substances that have been banned as pesticides are absorbed and transferred to agricultural products through the soil, acting as a new cause of nonconformity. Monitoring pesticide residues in farmland soil reveals that substances that have been banned as pesticides are still detected in these soils.

Previous studies monitoring organochlorine pesticides in the cultivation soil of facilities growing fruits and vegetables, such as strawberries, peppers, and melons, found that the frequency of detection of β-endosulfan and endosulfan sulfate at the survey sites was 11–35% and 38–51%, whereas the residual concentrations were 2.2–78.7 and 1.8–214.1 μg kg⁻¹, respectively [3,4]. In the cultivation soil of facilities cultivating leafy vegetables, such as perilla leaves and lettuce, the detection frequency of endosulfan sulfate was 11.9–48.1%, and the residual concentration was 22.0–123.8 μg kg⁻¹ [5]. These studies confirm that endosulfan has been continuously detected in farmland soil. In addition, based on the monitoring of residual pesticides in agricultural products conducted by the National Agricultural Products Quality Management Service from 2013 to 2018, the number of detections of endosulfan was 21–156 in Korea, and the residual concentration was 900–9000 μg kg⁻¹. Previous studies have also reported that endosulfan in the soil is absorbed and transferred to agricultural products [6,7,8].

Endosulfan is a mixture of stereoisomers α and β in a ratio of 7:3. In a slightly acidic-neutral soil environment, it is mainly converted into endosulfan sulfate and has a half-life of up to 2–3 years [9]. The Stockholm Convention was adopted in 2001 and enforced in May 2004, banning the manufacture, use, import, and export of persistent organic pollutants because they are bioaccumulated and have long-distance mobility. Endosulfan was classified as an endocrine disruptor at the 5th Stockholm Convention in 2009 in Genoa, Switzerland, owing to its negative effects on the central nervous system of mammals, including humans [10]. In Korea, it has been banned as a pesticide since 2011. It is currently designated as a restricted item for manufacturing, import, and export under the Toxic Chemicals Control Act of the Ministry of Environment [11,12]. Endosulfan’s human toxicity level is classified as Modern Hazardous by the World Health Organization (WHO). Exposure to a certain amount of endosulfan is considered harmful to the human body. The Joint FAO/WHO Meeting on Pesticide Residues (1998) set the non-toxic dose at 0.6 mg/kg bw/day, based on endosulfan-induced neurotoxicity, hepatotoxicity, renal toxicity, and weight loss, and a safety factor of 100 was applied to set the acceptable daily intake to 0–0.006 mg/kg bw/day, as evaluated previously [13].

Chemical substances in the soil are transferred to the human body mainly through ingesting food, which contains the chemical substance transferred from agricultural products. Previous studies have shown that endosulfan in the soil is absorbed and transferred to agricultural products [6,7,8]. Compared to the soil concentration of endosulfan, the crop concentration was reported to be <0.1% in grains, 1.3–4.7% in leafy vegetables, and 0.1–13.6% in fruits and vegetables [14,15,16]. Choi et al. [14] reported that carrot, a root vegetable, showed 28% of soil concentration, and a study by Hwang et al. [17] revealed that potatoes and carrots contained 26% of the initial soil concentration, showing a high level of transfer compared to other crops. The absorption and transfer of residues in the soil is known to increase as contact with the soil and contact time increase [6,18]. Accordingly, it is judged that root vegetables cultivated as perennials will have a high amount of endosulfan transfer in the soil, resulting in negative effects, such as making them unsuitable as raw material for agricultural products. Ginseng is one of the representative root vegetable crops in Korea. Ginseng (Panax ginseng C.A. Meyer) is a plant belonging to the Araliaceae family and has a growing period of 4–6 years, which is longer than that of other crops. Ginsenoside, a useful component found in ginseng, has many functional effects, such as liver protection, blood pressure control, and anticancer effects [19,20]. Therefore, ginseng is used to manufacture a variety of health-promoting functional foods and accounts for the highest production ratio among crops used as raw materials for health-promoting functional foods in Korea [18]. It is also one of the major agricultural items exported, currently being exported to 105 countries, accounting for 3.5% of total agricultural exports in 2021 [21,22]. In 2018, global ginseng production was approximately 86,223 tons, with China producing 50,164 tons, South Korea 23,265 tons, Canada 11,367 tons, the United States 1285 tons, Japan 30 tons, and other countries totaling 112 tons. The global value of ginseng production is estimated to be around $ 5900 million. The ginseng market is expected to expand further in the future due to the growing consumer interest in alternative medicine and health foods worldwide [23].

The residual endosulfan in ginseng cultivation soil is slowly reduced by photolysis and leaching as ginseng cultivation land is mulched and rain screens are installed. Therefore, it is judged that the risk of residual endosulfan in ginseng cultivation soil is high compared to other crop soils. In addition, as ginseng is a perennial root crop, there is a high possibility of endosulfan transfer in the soil. However, studies on the transfer of pollutant residues to crops for perennial root vegetables in the current soil are insufficient, and management plans are also lacking [12].

The safety management method for highly persistent substances such as endosulfan in the soil is to reduce them through physical methods, such as soil improvement, heating, and chemical treatment. However, the above-mentioned physical and chemical treatment methods are difficult to apply to farmland soil where crops are continuously grown because the methods require high treatment costs and do not allow crop cultivation [24,25]. Instead, methods for reducing absorption and transfer to crops using soil conditioners or inactivating soil materials can be applied to agricultural sites. Among soil conditioners, quicklime has an endosulfan-reducing effect. However, it is difficult to apply in the field because it increases the soil pH to over 10 and affects the cultivation of agricultural products [26]. Therefore, adsorbing endosulfan in the soil using biochar is considered the most realistic method for minimizing endosulfan absorption and transfer to ginseng.

Biochar is a carbon-rich by-product produced after the pyrolysis of biomass (agricultural by-products, wood, waste, etc.) heated in an anoxic environment. It does not affect the crop cultivation environment and is used for carbon sequestration, greenhouse gas reduction, and soil fertility improvement [27,28]. In addition, it also has a large surface area per unit volume and a porous surface, resulting in good water retention and high adsorption due to the large surface area of porosity [29]. It mitigates the effects of heat, drought, and salinity stress on crops. It also improves crop growth and productivity [30], increases biological N fixation in legumes [31,32], and facilitates carbon sequestration. However, the above-mentioned results are highly dependent on the type of biochar, the temperature at which the biochar is made, the biochar dose, and the soil type. A study evaluating the adsorption capacity of organic pollutants by treating soil with biochar reported that when the soil was treated with 2% (weight/weight) biochar, the adsorption capacity of the herbicide isoproturon was five times higher than that in untreated soil [30]. Tatarkova et al. [33] reported that treating soil with 1% biochar (weight/weight) increased 4-chloro-2-methylphenoxy acetic acid adsorption 2.53-fold. Yang et al. [34] reported that biochar in the soil increases the adsorption capacity of the soil, increasing the adsorption of diuron up to 80 times, thereby reducing the concentration of diuron in barnyard grass.

However, some studies have reported adverse effects on crop growth depending on the amount of biochar applied. Copley et al. [35] reported that the occurrence rate of Rhizoctonia solani, which can frequently occur in ginseng, increased by 40% among the experimental plants treated with biochar. Another study reported that biochar treatment could affect soil microbial activity by changing the soil environment [36]. Therefore, it is important to process an appropriate amount of biochar considering the soil characteristics in the cultivation environment.

In this study, we aimed to develop a safety management method for biochar that can be used in ginseng cultivation soil. To develop a safety management method for endosulfan in ginseng cultivation soil using biochar, ① we investigated the endosulfan reduction effect of different biochar treatments in soil; ② we evaluated the absorption, transfer, and reduction effect in ginseng; and ③ we evaluated the growth of above- and below-ground parts of ginseng and conducted germination surveys, according to the biochar treatments of the soil. Based on a comprehensive evaluation of these aspects, we propose a biochar treatment method that can be used in ginseng cultivation soil.

2. Materials and Methods

2.1. Chemicals and Reagents

The α-endosulfan, β-endosulfan, and endosulfan sulfate used in the experiment were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany), and the dichloromethane and acetone used for pretreatment and the working solution were purchased from J.T. Baker (Phillipsburg, NJ, USA) and Merck (Darmstadt, Germany).

2.2. Properties of Biochar and Soil Used in the Experiment

To prepare biochar of pH 10.2, EC 7.3 dS m⁻¹, bamboo and oak were dried in the shade to lower the moisture content to 15% in 15–20 °C, and it was further lowered to 10% using a dryer for 24 h. Following this, the dried material was placed in a biochar maker (DK1015, STI), maintained at 400 °C for 4 h, and then maintained at 600 °C for 4 h.

The physicochemical properties of the experimental soil were investigated after treatment with endosulfan and biochar according to the standards of agricultural science and technology research and analysis (ISBN: 9788948016499). Soil pH was measured using the shaking method [37], and organic matter content was measured using the Tyurin method [38] (

Table 1. Physicochemical properties of field soil used in the experiment.

Treatment	pH (1:5)	EC (dS m−1)	NO3	P2O5	K	Mg	Na	Ca	OM (g kg−1)
			(mg kg−1)		(cmol+ kg−1)
Control *	6.53	0.47	24.06	262.76	0.20	1.00	0.06	4.53	10.06
BC-0.1% **	6.70	0.51	29.01	266.33	0.20	0.86	0.05	4.07	17.72
BC-0.3%	6.82	0.41	24.18	299.43	0.27	0.88	0.06	4.20	28.86
BC-1.0%	7.37	0.33	16.71	257.29	0.36	0.80	0.06	4.40	59.06

* Control: endosulfan 1.0 mg kg⁻¹, ** BC: endosulfan 1.0 mg kg⁻¹ + biochar. EC: electrical conductivity, OM: organic matter

).

2.3. Field Experiment and Sampling

The experiment site was located at the Major Achievement and Prospect of Ginseng in Soi-myeon, Eumseong-gun, Chungcheongbuk-do, Republic of Korea (latitue 36.94321403023995, longitude 127.7545139107935 The average temperature of the experimental field area is 11 °C, with a high of 18 and a low of 5.1, and the average annual precipitation is 1220.8 mm (National Weather Service Weather Data Open Portal of Korea). To measure the soil retention of endosulfan and the degree of transfer to ginseng, endosulfan was applied to the soil at a concentration of 1.0 mg kg⁻¹ on 27 March 2018. After spraying 1.0 kg of endosulfan evenly on soil pre-mixed with topsoil in the treatment quadrat (four treatments with a total area of 48.6 × 8 m), it was homogenized by cultivating it ten times using a rotary cultivator. Biochar treatment was performed 5 d after endosulfan treatment to stabilize endosulfan in the soil. Biochar was applied at 0.1, 0.3, and 1.0% of soil weight.

After 2 d of biochar treatment, 63 ginseng seedlings (Panax ginseng C.A. Meyer, 7 × 9) were transplanted per treatment quadrat (1.8 × 1.8 m; 3.24 m²), and the transplanted ridges were covered with rice straw thatch and mulched with black vinyl to prevent moisture evaporation (Figure 1).

Soil sampling was performed at an average interval of 30 d in the first year (2018) and 60 d in the following year (2019) from the 3rd day after transplanting ginseng seedlings. For sampling the treatment quadrat, 1–2 cm of the topsoil was removed, and the soil was collected to a depth of 15 cm in each of the 27 compartments for each treatment quadrat and then mixed. The collected samples were dried in the shade for approximately 5–7 d and passed through a 2 mm sieve to ensure homogeneity (Agri-Environmental Resource Analysis Method. Jeollabuk-do Agricultural Research & Extension Services. Accessed on 8 August 2017 https://www.jbares.go.kr/board/download.jbares?boardId=BBS_0000001&menuCd=DOM_000000101001001000&paging=ok&startPage=47&dataSid=63772&command=update&fileSid=24466).

2.4. Ginseng Growth Evaluation by Treatment

A cool environment in the shade was created, and the temperature was maintained at around 15 °C in the early stage of growth and 20–25 °C in the late stage of growth, and 6~10 L per compartment was irrigated in the early and late stages of growth, and 15~18 L in midsummer. The quality and growth traits of ginseng were evaluated using the germination rate, weight, and growth length of the above-ground and below-ground parts for each treatment quadrat. The investigated traits were root length, body length, body diameter, weight, stem height, stem diameter, stem length, and leaf length (Figure 2). The germination rate was investigated by randomly selecting five compartments for each treatment quadrat six weeks after transplanting seedlings. At the time of collection, the number of individuals present above the ground surface as the above-ground part grew was measured for each treatment quadrat. Approximately 11 weeks after transplanting ginseng seedlings, the above-ground parts were randomly collected from five compartments for each treatment quadrat. Stem height was measured as the length from the ground part to the highest extended stem, leaf length was measured as the vertical length of the above-ground leaf, and stem diameter was measured as the width of the above-ground stem.

Growth of the below-ground part was evaluated by collecting crops from three randomly selected compartments for each treatment quadrat approximately six months after transplanting ginseng seedlings. Body diameter was measured as the diameter of the thickest part of the body of the root, body length as the length of the root body, root length as the length of the root under the body, and weight as the weight of the ginseng root body (Figure 2).

2.5. Residue Analysis of Endosulfan in Soil

The liquid–liquid extraction (LLE) method and the Florisil SPE cartridge (1 g 6 mL) method were used for the experiment.

An amount of 100 mL of acetone was added to 50 g of soil, shaking extraction was performed for 1 h, and the mixture was filtered under reduced pressure. The filtrate was concentrated under reduced pressure at 40 °C using a rotary vacuum concentrator (RV 10, IKA, Seoul, Korea) and transferred to a separatory funnel. Subsequently, 50 mL of distilled water, 10 mL of saturated NaCl, and 90 mL of dichloromethane were added, and the mixture was shaken at a constant speed. The dichloromethane layer was passed through sodium sulfate. Then, after nitrogen concentration in a 15-mL tube, 2 mL of acetone was added to the tube to dissolve the compound again. After activating the mixture with 6 mL of hexane using a Florisil cartridge (SPE, 1 g, 6 mL), 2 mL of the sample was loaded, and elution was performed with 8 mL of acetone/hexane mixture (1:9, v/v) and subjected to nitrogen concentration (EYELA MG-2200, 40 °C.). It was then re-dissolved in 2 mL of acetone and filtered through a syringe filter (0.45 μm), and the sample solution was analyzed using a GC-micro electron capture detector (μECD, Hewlett Packard, Palo Alto, CA, USA, HP5890). Analytical instrument conditions are listed in

Table 2. Analytical conditions for endosulfan in soils (GC-μECD, Hewlett Packard, Palo Alto, CA, USA, HP5890).

Items		Contents
Injection volume		1 μL
Injection mode		Split less
Inlet temperature		250 °C
Detector temperature		300 °C
Oven temperature		200 °C
Column		RTX-5 (30 m × 0.25 mm, 0.25 μm)
Gas (N2, mL min−1)		1.0
Oven ramp	°C Min−1	Next Temp. (°C)	Hold Min.	Run time
Initial		100	2	2
Ramp 1	10	220	2	16
Ramp 2	5	260	0	24
Ramp 3	10	300	8	36
Post run		0	0	36

.

2.6. Analysis of Endosulfan Residue in Ginseng

As with the soil experiments, the LLE method and Florisil SPE cartridge were used. The residual amounts of α-endosulfan, β-endosulfan, and endosulfan sulfate in the ginseng samples were purified using LLE and a Florisil cartridge (1 g, 6 mL) and then analyzed. An amount of 20 mL of ethyl acetate was added to 5 g of ginseng, followed by sonication for 10 min and shaking extraction for 1 h, followed by sonication for 10 min. After centrifugation (3000 rpm, 10 min, 4 °C), it was transferred to a separatory funnel and 10 mL of supernatant, 20 mL of distilled water, and 60 (20 × 3) mL of ethyl acetate were added to it, and the sample was shaken at a constant speed. The ethyl acetate layer was removed, concentrated under reduced pressure, and dissolved again by adding 1 mL of acetone/hexane 1:9 (v/v) solution. After activating the Florisil cartridge by adding 5 mL of hexane, 1 mL of the sample was loaded and eluted with 20 mL of acetone/hexane mixture (1:9, v/v), followed by nitrogen concentration (30 °C rotary evaporators). An amount of 1 mL of acetone was added to the dry matter to redissolve it, and this was then filtered through a syringe filter (0.22 μm), and the filtrate was analyzed using gas chromatography–mass spectrometry (GC-MSD; GCMS-QP2020, Shimadzu, Japan). Analytical instrument conditions are listed in

Table 3. Analytical conditions for endosulfan in ginseng (GC-MSD, Shimadzu GCMS-QP2020).

Instrument	GC
Column	RTX-5 ms (30 m × 0.25 mm, 0.25 μm)
Carrier gas	He (1.0 mL min−1)
Inlet temperature	270 °C
	Rate (°C min−1)				Temp. (°C)				Hold min
Oven	Initial				80				1
	8				220				10
	10				260				2
Instrument	MS
Ion source temp	RTX-5 ms (30 m × 0.25 mm, 0.25 μm)
Interface temp	He (1.0 mL min−1)
Solvent cut time	270 °C
Detector voltage	0 kV
Start time	End time			m/z			m/z			m/z
20.5	23.64			237			207			195
23.64	26.39			237			241			195
26.39	34			274			272			387
Endosulfan	α				β				sulfate
Retention time (min)	22.25				25.03				27.75
Target ion	237				195				274

.

2.7. Statistical Analysis: Repeated Measures ANOVA

Statistical analysis was conducted using the free and open-source statistical software, R project (version 4.0.0, R Core Team, 2020) and Jamovi (version 1.2.22.0, Stats Open Now, https://www.jamovi.org, accessed on 31 December 2020). ANOVA and repeated measures ANOVA TEST were used. ANOVA was used to evaluate the quality evaluation for each biochar treatment. Repeated measures ANOVA was used to compare the temporal change in residual soil endosulfan after biochar treatment. We used 3 replicates of data for soil and 5 replicates for crops. The repeated measures ANOVA compares means across one or more variables based on repeated observations. According to repeated measures ANOVA, the residual concentration of endosulfan, artificially treated in a ginseng-cultivated area, was decomposed over time.

2.8. Calculating the Half-Life of Endosulfan

Sigmaplot (version 14.0, Systat Software, Erkrath, Germany) and first-order kinetics (Equation (1)) were used to determine the half-life of endosulfan in the soil for each treatment. Ct indicates the endosulfan concentration at time t (mg kg⁻¹), C_O indicates the initial concentration (mg kg⁻¹), t is the day number, and k is the reduction rate constant [39]. The half-life of endosulfan was calculated as a value of ln 2/k.

$$Ct=C_o\times e^{-kt}.$$

(1)

3. Results

3.1. Limit of Quantitation and Recovery Rate Analysis

The linearity of the calibration curves of α-endosulfan, β-endosulfan, and endosulfan sulfate, which were the components tested for in this study, were analyzed. For this purpose, each standard was dissolved in acetone to prepare a standard solution with 0.002 to 1.0 mg L⁻¹ concentration and then analyzed using GC-ECD and GC-MSD. Linearity was confirmed by preparing a calibration curve for each concentration from the peak area of each component. Based on this, calibration curves and regression equations for each component were prepared. The R2 values of α-endosulfan, β-endosulfan, and endosulfan sulfate were higher than 0.9994, indicating good linearity of the calibration curve for each component. The validity of the test method for the quantitative analysis of α-endosulfan, β-endosulfan, and endosulfan sulfate was evaluated using the linearity of the calibration curve, limits of quantitation (LOQ), recovery rate, and relative standard deviation. To obtain the linearity of the calibration curve, α-endosulfan, β-endosulfan, and endosulfan sulfate were each dissolved in 100 mL of acetone, and a 100 mg L⁻¹ stock solution was prepared. This solution was diluted and mixed to prepare mixed standards at concentrations of 0.05, 0.1, 0.5, 1, and 5 mg L⁻¹. The linearity of the calibration curve was obtained as the average value obtained by repeatedly injecting these solutions five times. In addition, we determined the concentration at which the signal-to-noise ratio was 10 as the LOQ of α-endosulfan, β-endosulfan, and endosulfan sulfate. The recovery rate from soil and ginseng was 74.5–107.5, and the coefficient of variation was 4.9–9.5%.

3.2. Comparison of the Residual Dissipation Pattern of Endosulfan for Each Biochar Treatment

The traces of endosulfan for each biochar treatment in soil were investigated. Total endosulfan (sum of endosulfan-α, -β, sulfate) in soil not treated with biochar decreased from the initial level of 1.3 mg kg⁻¹ to 0.94 mg kg⁻¹ (73.1% of the initial residual amount) after 533 d of treatment. In the 0.1, 0.3, and 1.0% biochar treatment quadrats, total endosulfan was 0.79 mg kg⁻¹ (53.9%), 0.87 mg kg⁻¹ (73.6%), and 0.64 mg kg⁻¹ (52.11%), respectively (Figure 3). The eta squared (η²) value was calculated using the repeated ANOVA test to identify the main cause of decrease in the residual amount of total endosulfan in the soil considering time, biochar treatment, and the interaction between the two factors as potential causes (

Table 4. Repeated ANOVA, Test of Normality between treatments (endosulfan in soil).

	Subject	F-Value	p-Value	η2
α-endosulfan	Time	272.42	<0.001	0.709
	Time and Biochar	5.54	<0.001	0.043
	Biochar	461	<0.001	0.226
β-endosulfan	Time	91.90	<0.001	0.769
	Time and Biochar	3.33	<0.001	0.084
	Biochar	128	<0.001	0.078
Endosulfan sulfate	Time	106.6	<0.001	0.346
	Time and Biochar	11.7	<0.001	0.114
	Biochar	829	<0.001	0.513
Total endosulfan	Time	44.83	<0.001	0.594
	Time and Biochar	3.79	<0.001	0.151
	Biochar	105	<0.001	0.146

). The reduction effect of biochar was 0.15, and that of the interaction between the two factors was 0.15. The reduction effect over time was the largest at 0.59. Therefore, the decrease in the residual amount of total endosulfan in the soil was most affected by time.

The initial soil residues of α-endosulfan and β-endosulfan in all test treatment quadrats were 0.65–0.82, and 0.48–0.62 mg kg⁻¹, respectively. After 532 d of endosulfan treatment, the residual amount of α-endosulfan decreased to 0.16~0.26 mg kg⁻¹ and the residual amount of β-endosulfan decreased to 0.24~0.30 mg kg⁻¹ per treatment quadrat (Figure 4). After 532 d of endosulfan treatment, the reduction rates of α-endosulfan in the soil were 98, 84, 64, and 63%, whereas those of β-endosulfan were 50, 57, 37, and 50%, respectively, in the control and treatment quadrats treated with 0.1, 0.3, and 1.0% biochar.

After stabilization for 10 d following biochar treatment, the residual amount of endosulfan sulfate in the soil was 0.07, 0.04, 0.03, and 0.03 mg kg⁻¹ in the control and 0.1, 0.3, and 1.0% biochar-treated quadrats, respectively. To identify the factors affecting the residual amount of endosulfan sulfate in soil, η² was calculated using a repeated ANOVA test. The results revealed that the effect of biochar was 0.51, which was higher than that of time (0.35) and the interaction of both factors (0.11) (Table 4). In addition, when the pH is higher than 7, a lower amount of endosulfan is converted to endosulfan sulfate [40], and the conversion to endosulfan diol is dominant [41,42]. In this study, when 1.0% biochar was added to the soil, the soil pH increased to 7.4, suggesting that the conversion to endosulfan sulfate was reduced (The biochar 0.3% treatment residue data is same as reference [43]).

At 66 days in each treatment, endosulfan sulfate conversion decreased from 28.2% in the control treatment to 16.8, 8.6, and 3.9% in the 0.1, 0.3, and 1.0% biochar treatments, respectively (Figure 5), and the rate of endosulfan sulfate production decreased over time in each treatment as the biochar content increased (Figure 6). The conversion rate of endosulfan sulfate according to biochar treatment in soil remained at approximately 68.8% of total endosulfan. However, total endosulfan residues were 47.2, 37.8, and 21.7%, respectively, for each quadrat treated with 0.1, 0.3, and 1.0% biochar. Therefore, the conversion rate of endosulfan sulfate decreased as the biochar throughput increased. The formation of endosulfan sulfate in the soil environment over time may have contributed to the lack of significant results according to the treatment quadrat.

α-Endosulfan showed a half-life of 87.4 d in the control treatment and 72.4, 67.0, and 64.6 d in the biochar 0.1, 0.3, and 1.0% treatments, respectively, indicating a reduction effect according to biochar treatment; however, β-endosulfan and total endosulfan showed no reduction effect (63.5–66.9 d and 50.4–56.3 d, respectively).

3.3. Reduction Effect of Different Biochar Treatments on Endosulfan Uptake from Soil to Ginseng

The effect of the reduction in the absorption and transfer of endosulfan to ginseng for different biochar treatments in the soil was investigated. Ginseng was harvested after cultivating two-year-old ginseng seedlings for six months after transplantation. The total endosulfan concentration of ginseng harvested from untreated quadrat soil was 2.30 mg kg⁻¹, and those of quadrats treated with 0.1, 0.3, and 1.0% biochar were 1.31, 0.79, and 0.20 mg kg⁻¹, respectively. Therefore, in ginseng cultivated under different biochar treatments, the reduction effect of endosulfan absorption concentration in ginseng increased by 51.0, 70.4, and 92.5% compared to that of ginseng cultivated in the untreated quadrats (Figure 5). The residual amounts of α-endosulfan in ginseng were 0.50, 0.19, 0.14, and 0.07 mg kg⁻¹, respectively. Treatment with biochar reduced absorption and transfer by 62–86% compared to the control treatment quadrat; this percentage was 55–92% for β-endosulfan with residual amounts of 0.53, 0.24, 0.16, and 0.04 mg kg⁻¹, respectively. The amount of endosulfan sulfate in ginseng was 1.63 mg kg⁻¹ in untreated quadrats. Quadrats treated with 0.1, 0.3, and 1.0% biochar showed residual amounts of 0.87, 0.49, and 0.09 mg kg⁻¹, respectively, showing a reduction effect of approximately 47–95% (Figure 7).

BCF (BCF, concentration in ginseng/concentration in soil) was used to determine the extent of absorption and transfer of endosulfan in the soil. BCF was calculated using the initial residual concentration in soil and the residual concentration in harvested ginseng. The BCF in the control treatment quadrat was 2.01. In the 0.1, 0.3, and 1.0% biochar treatment quadrats, the BCF was 0.89, 0.66, and 0.16, respectively, showing a decrease of 56.9–92.03% compared to the control treatment quadrat (Figure 8,

Table 5. Concentration of endosulfan in ginseng with BCF (bioconcentration factor), SE–Standard Error.

	Ginseng (SE)			BCF (SE)
	α-Endosulfan	β-Endosulfan	Endosulfan-sulfate	p < 0.001
Control	0.50 (0.04)	0.53 (0.04)	1.63 (0.12)	2.01 (0.38)
Biochar 0.1%	0.19 (0.01)	0.24 (0.01)	0.87 (0.05)	0.89 (0.14)
Biochar 0.3%	0.14 (0.01)	0.16 (0.01)	0.49 (0.01)	0.66 (0.03)
Biochar 1.0%	0.07 (< 0.01)	0.04 (< 0.01)	0.09 (< 0.01)	0.16 (0.01)

).

3.4. Quality Evaluation and Germination of Ginseng by Biochar Treatment

Table 6. Comparison of ginseng quality evaluation by biochar treatment (average of each treatment with standard error).

Treatment	Root Length (cm)	Body Length (cm)	Body Diameter (mm)	Weight (g)	Stem Height (cm)	Stem Diameter (mm)	Leaf Length (cm)
Control	16.852 (1.17)	12.162 (0.55)	8.155 (0.21)	3.711 (0.25)	6.908 (0.19)	2.093 (0.07)	7.418 (0.17)
En + BC 0.1%	17.383 (0.97)	12.212 (0.33)	9.181 (0.25)	5.263 (0.35)	6.634 (0.32)	1.994 (0.04)	7.240 (0.14)
En + BC 0.3%	17.683 (1.07)	11.533 (0.46)	8.851 (0.38)	4.578 (0.43)	5.920 (0.19)	1.940 (0.03)	7.178 (0.19)
En + BC 1.0%	17.908 (0.68)	12.463 (0.42)	8.408 (0.23)	4.347 (0.17)	6.360 (0.25)	2.073 (0.03)	6.998 (0.16)
F-value	0.368	0.397	2.056	2.654	1.365	1.144	0.355
p-value	0.826	0.807	0.162	0.096	0.281	0.365	0.838

shows the post-harvest appearance of two-year-old ginseng (Figure 9) and the quality evaluation results for each trait. In addition, the germination rate observed during the optimal growth period of ginseng was confirmed for each treatment quadrat. Ginseng harvested from soil in the control and 0.1, 0.3, 1.0% biochar-treated quadrats weighed 3.5–5 g, the stem length was 5.4–7.8 cm, stem diameter was 1.8–2.2 mm, leaf length was 6.5–8 cm, root length was 13.7–19.8 cm, fuselage length was 10.4–13.3 cm, and fuselage diameter was 7.7–9.6 mm. No significant difference was observed between treatment quadrats (p > 0.05).

However, unlike the quality of harvested ginseng, the germination rate tended to decrease in quadrats treated with more than 0.3% biochar (p < 0.05). In the growth evaluation of ginseng, the germination rate of two-year-old ginseng was higher than 80% in the control and 0.1% biochar treatment quadrats, 59.4% in the 0.3% biochar treatment quadrats, and 56% in the 1.0% biochar treatment quadrats. The germination rate was significantly lower in quadrats treated with more than 0.3% biochar. In ginseng-cultivated soil, pH indicates the nutrient supply capacity, and the optimum pH is 5.0–6.5. When the soil was treated with 0.3% and 1.0% biochar, the pH increased to approximately 6.8 and 7.4, which was outside the optimal pH range. In addition, one of the crucial factors that can affect ginseng germination rate is the soil’s organic matter content. In this study, the control and 0.1% biochar-treated quadrats contained 10 to 30 g kg⁻¹ of soil organic matter content, which was an appropriate level.

4. Discussion

In this study, the concentration of endosulfan sulfate increased over time in the treatment area where biochar was not applied, and biochar treatment inhibited the conversion of metabolite endosulfan sulfate by adsorption with biochar in the soil. As a result, a significant reduction in the concentration of endosulfan sulfate was achieved through biochar treatment. Furthermore, the concentration of α-endosulfan decreased over time, along with the concentration of endosulfan sulfate, and soil pH increased from 6.5 to 7.4 when the soil was treated with 1.0% biochar, promoting the decomposition of α-endosulfan, which may have affected the reduction of α- and β-endosulfan. β-Endosulfan in the soil did not depend on pH. However, the decomposition rate of α-endosulfan decreased with increasing pH [23]. An alternative explanation for this may be found in the study by Walse et al. [44], who reported that α-endosulfan is more readily converted to endosulfan sulfate compared to β-endosulfan. Therefore, the differences in degradation rates observed in the current study may have occurred because residual α-endosulfan was more readily converted to endosulfan sulfate in the soil environment compared to β-endosulfan. The distribution coefficient (Kd) of β-endosulfan in the soil is higher than that of α-endosulfan [45], which suggests that β-endosulfan is preferentially adsorbed on clay and organic surfaces and remains there.

Endosulfan sulfate is one of the major metabolites in the agricultural use of endosulfan, which is mainly formed in weakly acidic and neutral soil environments [46] and is more difficult to decompose and more persistent than the parent compounds [47,48,49,50,51]. Further, endosulfan sulfate is associated with high reproductive toxicity, such as a decrease in the hatching rate of zebrafish [52]; thus, further research on endosulfan toxicity and behavior in the environment is required. Endosulfan sulfate affects the cytochrome P450 hormone in mammals [53,54]. Endosulfan sulfate can cause an increase in MDA levels in living organisms, which can lead to biologically harmful effects of disturbance in organs. [55]. In the present study, the conversion of endosulfan sulfate in soil was suppressed through biochar treatment, and at the same time the endosulfan concentration transferred to ginseng was reduced. This was due to the irreversible adsorption effect in the biochar treatment, which led to a low desorption effect [56], which is believed to have reduced the bio-use rate of pesticides. Such strategies may help create a sustainable agricultural environment. Further, the adsorption capacity of biochar is expected to result in more suitable soil conditions for the subsequent crop in agricultural systems where the organic substances dildrin, heptachlor, and chlordein occur with high Koc values.

Biochar has high adsorption capacity for organic pollutants. Wang et al. [57] report-ed that heat treatment of biochar at 700 °C and 300 °C increased the adsorption coefficient (Kd) of soil by 63-fold and 2.9-fold, respectively. As a result, they reported that the adsorption of terbuthylazine could be increased by 2.9 times through biochar heat-treated at 700 °C. When a pesticide is adsorbed on biochar, its bioavailability is reduced because it is not a bioavailable fraction, thus inhibiting the uptake and transfer of the pesticide to plants [34,58,59]. In addition, biochar is porous and has strong adsorption properties. In addition, pesticides in the soil can be adsorbed through mechanisms such as covalent bonding of soil organic matter and entrapment in micropores [60]. As a result, it is likely that the effect of reducing absorption and transfer increased as the amount of biochar applied increased.

Bioaccumulation data are expressed using a bioconcentration factor (BCF, concentration in ginseng/concentration in soil), translocation factor (TF, transfer from roots to edible parts), and bioaccumulation factor (BAF, coefficient considering all bioconcentration, surface absorption, and feeding) [61,62]. In this study, as the root in contact with the soil is the main edible part of ginseng, BCF was used to determine the extent of absorption and transfer of endosulfan in the soil.

There was no significant difference in the quality effect of two-year-old ginseng, according to biochar treatment in soil in all treatment groups (p > 0.05). The results suggest that treatment of ginseng cultivation soil with 1% biochar does not affect ginseng growth. As a soil conditioner, biochar has the effect of improving water retention and nutrition [63]; it improves the physicochemical properties of soil treated with biochar [41] and increases fertilizer application efficiency and crop production by effectively maintaining the level of soil organic matter [64,65,66,67]. In addition, biochar has an excellent ability to adsorb organic and inorganic contaminants owing to its high surface area carbon structure, which is believed to inhibit conversion to organochlorine pesticides [36,56]. However, in this study, biochar treatment did not significantly improve the quality of ginseng, as measured through different growth parameters. Because crop quality is affected by climate and soil physicochemical properties, the results of this study differ from those of previous studies [68]. However, when the soil was treated with 0.3% biochar or more, it exceeded the appropriate range and increased to a maximum of approximately 59 g kg⁻¹. Optimum organic matter content can improve soil and increase productivity. However, excessive organic matter content in ginseng-cultivated soil can cause physiological disorders owing to an imbalance in the soil environment [69]. As a soil conditioner, biochar may contain small amounts of phenolic compounds and heavy metals harmful to plants. These compounds can affect seed germination and seedling growth [70]. Some studies have shown that when the amount of biochar applied to the soil is more than 3%, it can negatively affect plant growth and accelerate plant disease [71,72,73]. Therefore, it is necessary to maintain an appropriate level of biochar. In this study, as the amount of biochar was increased to 0.1, 0.3, and 1.0%, the soil pH increased by 3, 4, and 14%, and the organic matter content increased by 0.8, 1.9, and 4.9 times, respectively, compared to the control. The average pH of agricultural soil in Korea is 6.2, and the organic matter content is 23.3 g kg⁻¹ [74]. Considering this, 0.1% biochar application, which does not affect the growth environment, is appropriate. However, the physicochemical characteristics of the soil environment differ depending on the region, cultivation period, and type [75]. Therefore, it is necessary to determine how much biochar should be added after checking the physical and chemical properties of the soil used for ginseng cultivation.

5. Conclusions

This study aimed to develop an endosulfan safety management method using biochar that can be applied to ginseng cultivation soil. Specifically, the effect of biochar treatment on endosulfan reduction in soil and absorption/transfer and reduction in ginseng was evaluated after biochar treatment. In addition, we evaluated the growth of the above- and below-ground plant parts before and after treatment with biochar and conducted germination research.

The initial residual amount of total endosulfan (sum of endosulfan-α, β, sulfate) in the soil was 1.3 mg kg⁻¹, which was reduced to 73.1% of the initial residual amount after 533 d of treatment. In the treatment quadrat treated with 0.1, 0.3, and 1.0% biochar, the endosulfan levels were 53.9, 73.6, and 52.1%, respectively. To identify the factor for the decrease in endosulfan in the soil, we calculated the η² value using two-way ANOVA, and the time factor was the highest at 0.594. However, biochar had the highest η² value of 0.513 as the main factor that reduced the conversion to endosulfan sulfate. Soil treatment with biochar did not affect total endosulfan reduction, but reduced endosulfan sulfate conversion.

When 1.0% biochar was applied in ginseng cultivation soil, α-endosulfan and β-Endosulfan reduced to 86% and 92% compared to the residual amount in ginseng grown in the control, demonstrating the reduction in the absorption and transfer of endosulfan. In ginseng grown in soil treated with 0.1, 0.3, and 1.0% biochar, the reduction effect of endosulfan sulfate increased from 47 to 95% with increasing treatment amount compared to the control. When soil is treated with biochar, the conversion of endosulfan sulfate in the soil is inhibited; therefore, the residual amount in biochar-treated soil is lower than that in control soil, and the material is strongly adsorbed to biochar and deactivated.

Previous studies have demonstrated that soil treatment with biochar helps crop growth by improving soil fertility; however, excessive use has a negative effect on crop growth. Therefore, optimal conditions that do not affect ginseng cultivation and reduce endosulfan absorption/transfer must be established for field application. Even when the soil was treated with up to 1.0% biochar, there was no significant difference in ginseng weight, root and trunk length, trunk diameter and stem diameter, and length compared to the control (p > 0.05). However, the ginseng germination rate was 59% and 56% in the quadrat treated with 0.3% and 1.0% biochar, respectively, which was lower than the germination rate of over 80% in the control and 0.1% biochar treatments. Optimal soil pH and organic matter content for ginseng cultivation are 5.0–6.6 and 10–30 g kg⁻¹. However, when biochar was applied at concentrations of 0.3% or more, we judged that the two factors increased and exceeded the appropriate range, affecting the germination rate.

It is necessary to consider the absorption/transfer reduction effect and crop growth evaluation to utilize the endosulfan safety management method for ginseng cultivation soil in the field. A proportion of 0.1% biochar is appropriate considering the domestic soil pH and organic matter content and the effect of reducing the absorption and migration of biochar obtained from the results of this study. However, it is necessary to determine the amount of biochar to be added after examining the physical and chemical properties of the soil used for ginseng cultivation.

Ginseng was sown in the first year and transplanted into the cultivation soil in the second year. The germination rate of ginseng was investigated in the second year. The germination rate is an important part of ginseng growth because it is linked to the quality and yield of ginseng in the future. In general, the growth period of ginseng is 4 to 6 years. Ginseng exhibits growth characteristics such as active root development and very rapid nutrient absorption compared to other plants. Therefore, additional studies investigating the effects of various components in the soil on ginseng are required.