Anthracene Absorption and Concentration Dynamics in Radishes

Abstract

This study examined the absorption and concentration of anthracene (AN) in the garden radish. Experiments were conducted to grow radishes from the sowing stage in soil contaminated with AN and to grow radishes in soil contaminated with AN following maturity (27 days after sowing). Regarding growth in the AN-containing soil from sowing onward, the AN concentrations during the growing period in both the soil and radishes decreased exponentially over time, albeit with a time lag. Regarding growth in the AN-containing soil after maturity, the AN concentrations in the roots and leaves had a higher density than those in the stems. In these experiments, positive relationships between the AN levels in the soil and radishes were observed. It was inferred that the greater the degree of soil contamination, the greater the effect was on the amount of AN assimilated by radishes. The concentration ratio (radish-to-soil; Cr/Cs) decreased exponentially with the number of days following AN application. It was interpreted that AN was significantly absorbed in the early stages of radish growth and some of the AN returned to the soil as the growth period progressed. According to the results regarding the change over time in the Cr/Cs content in radish parts, it is suggested that soil AN was well absorbed through the roots to circulate within the organism, and it is preferentially distributed and accumulated in the roots and leaves, which have high organic matter content.

1. Introduction

Anthracene (hereinafter referred to as “AN”) is a polycyclic aromatic hydrocarbon that is used as an insecticide, gasoline stabilizer, triplet sensitizer, and quenching agent. As AN is generated unintentionally by the burning of the fossil fuels such as petroleum and coal, it is discharged into the environment from numerous emission sources, including automobiles, incinerators, and factories [1,2,3,4,5]. Accordingly, AN has been detected in air, water, and other environmental media around the world [6,7,8,9,10,11,12,13,14,15,16,17,18,19]. In an evaluation by the International Agency for Research on Cancer (IARC), AN was categorized as a compound that cannot be evaluated with respect to carcinogenicity in humans (Group 3) [20]. However, due to its persistence, accumulation in ecosystems, and toxicity, it was included in the first list of substances of very high concern (SVHC) released by the European Chemicals Agency in 2008, and it can be considered a harmful chemical substance of note [21]. The accurate calculation of the health risk of AN requires the collection of information on the exposure level; the accumulation of data on its concentration in environmental media including water, air, and food; and the consideration of the substance’s environmental dynamics.

The present study focused on the radish vegetable (Raphanus sativus). This member of the Brassicaceae family is native to Europe. Radishes, of which R. sativus is one type, are among the most popular vegetables in Japan. The plant can be harvested approximately 1 month after sowing when grown at a temperature of approximately 20 °C. In large specimens, the leaves grow to a height of approximately 30 cm, and the root develops into a red, spherical, tuberous root with a diameter of approximately 3 cm. The radish was selected for this study, because the time until harvest is short, and its growth management is relatively straightforward. Previous studies have investigated the uptake and distribution of micropollutants, as well as pesticides, heavy metals, and pharmaceuticals in radishes [22,23,24,25,26,27,28,29,30]. However, limited information is available on the uptake pathways and accumulation of AN by vegetables. To construct an AN exposure assessment system, this study aimed to evaluate its absorption and concentration properties in radishes grown in contaminated soil and specifically the temporal variation in its absorption and the dynamics of its accumulation in radish tissue.

2. Materials and Methods

2.1. Chemicals

The Polynuclear Aromatic Hydrocarbons Mix (CRM48905) used for the determination of AN was purchased from Sigma-Aldrich (St. Louis, MO, USA). The Three Internal Standards Mixture Solution (091-05791) used for the determination of AN-d10, hexane for pesticide residue and polychlorinated biphenyl analysis (084-04761) and sodium sulfate (197-03345) were purchased from the FUJIFILM Wako Pure Chemical Corporation (Osaka-shi, Osaka-fu, Japan). Hexane was used for the preparation of standards, the cleaning of laboratory glassware and disposables before analysis, and the extraction of AN and AN-d10 from samples. Sodium sulfate was used for dehydration because water present in a sample can interfere with the measurement equipment. The water used in this study was produced by using an Arium^® water purification system (Sartorius AG, Göttingen, Germany), while tap water was used for the irrigation of the growth medium.

2.2. Plant Material and Radish Growth

Radish seeds (Z57-10-00181) were purchased from the Tohoku Seed Co. (Utsunomiya, Tochigi, Japan). A young radish seedling, collected 6 days after sowing, and a mature radish plant, obtained 28 days after sowing, are shown in Figure 1a,b. The total length of a young radish seedling was approximately 7 cm, and its total weight was approximately 0.2 g. The total length of the largest mature radish plant exceeded 30 cm, and its total weight was more than 20 g. The length of the mature radish root was approximately 5 cm, and the width of the bulbous portion was approximately 3 cm. Commercial potting soil (4522831033355) was purchased from Kohnan (Osaka-shi, Osaka-fu, Japan). The soil was a blend of materials based on peat moss, coconut fiber, vermiculite, and perlite. Fertilizer (4905832360109) was purchased from Tosho (Yaizu, Shizuoka, Japan). The fertilizer contained nitrogen, phosphorus, potassium, magnesium, and calcium. A potting stone (G515042F) was purchased from Iris Ohyama (Sendai, Miyagi, Japan). The potting stone was composed of sterile pumice that had undergone heat sterilization treatment.

The radishes were grown in planters in a room with abundant sunlight from windows. The planters used were 45 cm in width, 30 cm in length, and 18 cm in depth. As shown in Figure 2, approximately 400 g of potting stones were laid evenly on the bottom of a planter, which was then covered with garden-use potting soil to a depth of approximately 8 cm (approximately 400 g). The planters were further prepared using potting soil mixed with fertilizer and AN, with the soil laid to a depth of approximately 16 cm. The masses of the potting soil and the fertilizer were approximately 1600 g and 15 g. The amount of anthracene added is described in the next section. After the planters were readied, approximately 2 L of water was sprinkled into the planters, and approximately 60 radish seeds were buried in two rows. The radishes were grown with 300 mL to 500 mL of water, sprinkled nearly every day. When the air temperature was high, the windows were opened to adjust the temperature inside the growing room, except when it was raining or windy.

2.3. Experimental Conditions

Experiments were conducted to grow radishes from the sowing stage in soil contaminated with AN and to grow radishes in soil contaminated with AN following maturity (27 days after sowing). The experiments were performed using four types of planters, as shown in

Table 1. Radish growing conditions.

Planter No.	Growing Period	Air (°C)	Soil (°C)	AN Amount
P1	3 June–1 July 2019	26.7	25.9	0.05 g
P2	3 June–1 July 2019	26.7	26.9	0.10 g
P3	2 July–30 July 2019	28.2	28.9	0.20 g
P4	1 June–22 July 2019	27.6	26.5	See Section 2.3

Note: Air (°C)—average of temperatures measured at the growing site during the radish growing period (during sprinkling of water, between 0800 and 1000 each day). Soil (°C)—average of five measurements (center and four corners of planter; measured during sprinkling of water).

. The study of growth in contaminated soil from sowing onward was conducted using planters P1 and P2 from 3 June to 1 July 2019 and using planter P3 from 2 July to 30 July 2019. The study of growth in contaminated soil from maturity onward was conducted using planter P4 from 1 June to 22 July 2019. The mean air temperature during the growing period ranged from 26.7 °C to 28.2 °C, while the mean soil temperature was between 25.9 °C and 28.9 °C. The AN amount added to the potting soil laid between 8 cm and 16 cm (1600 g) was 0.05 g in P1, 0.10 g in P2, and 0.20 g in P3. In P4, which was used to study growth in contaminated soil after maturity, a suspension of AN (1.6 mg to 4.5 mg of AN added to approximately 20 mL of pure water) was sprayed in water around the roots 27 days after sowing, after which growth was continued.

2.4. Sampling of Specimens

For the radishes grown in contaminated soil from sowing onward, radishes and soil were collected five times in P1, P2, and P3 immediately after sowing and at 6 days, 14 days, 21 days, and 28 days after sowing. From these, 20 to 27 plant seeds were collected 6 days after sowing, 10 to 17 plants were collected 14 days after sowing, 5 to 6 plants were collected 21 days after sowing, and 3 plants were collected 28 days after sowing. Soil specimens with a diameter of approximately 3 cm and a depth of approximately 3 cm were collected from five locations (the center of the planter and near the four corners). The specimens were collected and mixed to create test samples.

In P4, which was used to study growth in contaminated soil after maturity, the plants and soil were collected six times: before the application of AN and 1 day, 4 days, 11 days, 19 days, and 24 days after application (27 days, 28 days, 31 days, 38 days, 46 days, and 51 days after sowing). One radish was collected on each of these days. Soil with a diameter of approximately 5 cm and at a depth of approximately 5 cm surrounding the collected radish was collected and mixed to prepare a test sample.

2.5. Analysis of Anthracene

The radish samples were thoroughly washed with water, wiped, and weighed using an electronic scale. Each sample was then cut into pieces several millimeters in size using scissors. The pieces were placed in a cylindrical cellulose filter paper (Whatman, UK) and were weighed again to determine the exact sample mass for ultrasonic extraction using hexane. At 6 days after sowing in P1, P2, and P3, the radishes were small and could not be divided into roots, stems, and leaves; thus, the 20- to 27-day plants were combined, chopped into pieces several millimeters in size, and mixed, after which a portion of the sample was placed in the cylindrical cellulose filter paper and weighed. As the plants in P1, P2, and P3 on days other than 6 days after sowing, and the plants in P4, had grown large, these were separated into roots, stems, and leaves, each of which were chopped into pieces several millimeters in size and mixed thoroughly. A portion of the sample of each part was placed in a cylindrical cellulose filter paper and weighed. The soil samples were also placed in cylindrical cellulose filter papers and weighed.

The cylindrical cellulose filter papers containing the samples were placed in 260 mL bottles, into which 150 mL of hexane was added; after this, ultrasonic extraction was performed for 15 min. In order to remove the water from the samples, which had a negative impact on GC/MS, anhydrous sodium sulfate was added to the extract liquid. After dehydration for 30 min, the solution was concentrated to approximately 2 mL using a rotary evaporator. This concentrated solution was combined with the solution obtained by rinsing the rotary evaporator device with hexane; anhydrous sodium sulfate was added to the combination, and the mixture was dehydrated for 30 min. The dehydrated solution was passed through a syringe filter (Whatman Puradisc 25 TF; GE Healthcare Bio-Sciences, Piscataway, NJ, USA) and was concentrated to 1.5 mL under a stream of nitrogen. Next, 0.2 mL of internal standard solution (100-fold diluted solution of hexane, 3 Internal Standards Mixture Solution) was added. The mixture was accurately adjusted to 2.0 mL using hexane, and a sample solution for the GC/MS measurement was prepared.

The GC/MS device used for measurement was the 5975B inert XL E/CI MSD (Agilent Technologies, Santa Clara, CA, USA). The capillary column used was the HP-5MS (30 m × 0.25 mm × 0.25 μm). The injection port temperature was 250 °C, with a splitless injection used. The injection volume was 2 μL; the column temperature rise protocol was 70 °C (1.5 min) → 20 °C/min → 180 °C (0 min) → 5 °C/min → 290 °C (10 min). The carrier gas was helium, and the interface temperature was 230 °C. A mass spectrometer was used to perform measurements under electron impact ionization mode under a 70 eV ionizing voltage. For AN, mass numbers 178.1 and 176.1 were selected; for the internal standard substance AN-d10, mass numbers 188.2 and 189.2 were selected for use in the qualitative evaluation. The quantitative evaluation was performed using the internal standard calibration method, using the mass numbers with the highest sensitivity (178.1 for AN and 188.2 for AN-d10). Standard solutions were produced through mixing and through dilution with hexane as appropriate, using the Polynuclear Aromatic Hydrocarbons Mix for AN and the 3 Internal Standards Mixture Solution for AN-d10.

3. Results

3.1. Results for Radishes Grown from Sowing Onward in Soil Contaminated with Anthracene

Table 2. AN concentration in soil and radishes in experiments under growth in contaminated soil from sowing onward.

Planter No.	AN	Sample Type	Elapsed Time after Seeding
			0 Days	6 Days	14 Days	21 Days	28 Days
P1	0.05 g	Soil	12,500	8040	8840	7980	7140
		Radish		1170	214	55.4	38.2
				(5.16 g) 23 *	(14.1 g) 17 *	(24.6 g) 5 *	(45.2 g) 3 *
		Root			2030	374	31.8
					(1.37 g)	(2.57 g)	(17.3 g)
		Stem			2.28	1.29	47.2
					(5.32 g)	(9.86 g)	(13.1 g)
		Leaf			33.2	32.3	37.8
					(7.43 g)	(12.2 g)	(14.8 g)
P2	0.10 g	Soil	23,300	11,900	10,300	9020	8770
		Radish		1620	547	200	122
				(6.29 g) 27 *	(13.2 g) 14 *	(26.6 g) 6 *	(48.0 g) 3 *
		Root			4410	2580	305
					(1.33 g)	(1.37 g)	(15.6 g)
		Stem			22.7	41.2	52.9
					(4.72 g)	(11.6 g)	(15.1 g)
		Leaf			174	96.5	18.3
					(7.18 g)	(13.6 g)	(17.3 g)
P3	0.20 g	Soil	36,300	16,000	13,800	12,000	11,300
		Radish		2160	207	337	189
				(3.87 g) 20 *	(9.09 g) 10 *	(19.4 g) 6 *	(26.3 g) 3 *
		Root			853	3630	250
					(0.88 g)	(1.20 g)	(6.38 g)
		Stem			16.8	24.9	2.05
					(3.31 g)	(8.25 g)	(7.40 g)
		Leaf			219	198	268
					(4.90 g)	(10.0 g)	(12.5 g)

Note: Units are ng/g-ww, unless otherwise noted; ng/g-ww means ng of AN per 1 g of sample wet weight. Blank cells indicate that there was no measurement at this point in time. AN concentrations and weights were determined for each part of a plant for root, stem, and leaf samples. “Radish” denotes measured AN data for the whole plant; the concentration was multiplied by the weight for each part, the products added together, and the total divided by the sum of the weight. The upper numerical data in each cell are concentrations (ng/g-ww); the middle numerical data in the parentheses are masses (g); the lower values with asterisks (*) are the numbers of radish samples measured. The upper values for root, stem, and leaf measurements are concentrations (ng/g-ww); the lower values in the parentheses are masses (g).

shows the detailed results of the experiment (P1, P2, and P3), in which radishes were grown in AN-contaminated soil from sowing onward. Figure 3a shows the AN concentration in the contaminated soil, and Figure 3b shows the AN concentration in the radishes over time. Quantities of 0.05 g, 0.10 g, and 0.20 g of AN were added to the topsoil in P1, P2, and P3, respectively. The AN concentration in collected soil that had been sprinkled with approximately 2 L of water immediately after sowing was 12,500 ng/g-ww in P1, 23,300 ng/g-ww in P2, and 36,300 ng/g-ww in P3. As shown in the figure, the concentration subsequently decreased exponentially. The AN concentration in the soil collected 28 days after sowing was 7140 ng/g-ww in P1, 8770 ng/g-ww in P2, and 11,300 ng/g-ww in P3. The AN concentration in radishes collected 6 days after sowing was 1170 ng/g-ww in P1, 1620 ng/g-ww in P2, and 2160 ng/g-ww in P3. The concentration subsequently decreased exponentially 28 days after sowing to 38.2 ng/g-ww in P1, 122 ng/g-ww in P2, and 189 ng/g-ww in P3.

3.2. Results for Radishes Grown after Maturity in Soil Contaminated with Anthracene

Table 3. AN concentration in soil and radishes under late exposure experiments.

Planter No.	Sample Type	Day (after Seeding)
		0 * (27)	1 * (28)	4 * (31)	11 * (38)	19 * (46)	24 * (51)
P4	Soil	N.D.	17,900	22,100	5670	14,400	9560
	Radish	N.D.	2180	2270	879	967	342
			(17.1 g)	(13.9 g)	(34.1 g)	(35.6 g)	(52.8 g)
	Root	N.D.	3470	228	2230	1820	742
			(4.92 g)	(4.03 g)	(11.0 g)	(16.3 g)	(22.4 g)
	Stem	N.D.	230	52.2	182	143	39.5
			(5.80 g)	(4.55 g)	(10.2 g)	(9.87 g)	(15.7 g)
	Leaf	N.D.	2960	5750	283	356	55.9
			(6.34 g)	(5.28 g)	(13.0 g)	(9.46 g)	(14.7 g)

Note: Units are ng/g-ww, unless otherwise noted. N.D. indicates ‘not detected’. Asterisks (*) show the number of days after AN was buried; the values in parentheses denote the number of days after seeding. AN concentrations and weights were measured for each part of a plant for root, stem, and leaf samples. “Radish” denotes measured AN data for the whole plant; the concentration was multiplied by the weight for each part, the products added together, and the total divided by the sum of the weight. The upper values in each cell are concentrations (ng/g-ww); the lower values in the parentheses are masses (g).

shows the detailed results of the experiment (P4) in which radishes were grown in AN-contaminated soil after maturity. The conditions included an average outdoor air temperature of 25.3 °C during the growing period, with AN not added at the time of sowing, and a suspension of AN sprinkled around the roots of the radishes 27 days after sowing. The concentration in the soil was the highest 4 days after the application of AN (31 days after sowing), at 22,100 ng/g-ww, and it was the lowest 11 days after the application of AN (38 days after sowing), at 5670 ng/g-ww. Although there were some fluctuations due to AN suspensions of differing content being sprinkled around the roots, the concentration generally showed a decreasing trend.

Similar to the concentration in the soil, the concentration in the radishes tended to decrease over time following the addition of AN, with the highest concentration observed 4 days after the application of AN, at 2270 ng/g-ww, and the lowest concentration observed 24 days after the application of AN, at 342 ng/g-ww. The trends in concentration differed according to the part of the radish examined. The concentration in the roots and stems was the highest 1 day after the application of AN, at 3470 ng/g-ww and 230 ng/g-ww, respectively. The concentration in the leaves was the highest 4 days after the application of AN, at 5750 ng/g-ww. The concentration was the lowest in the roots 4 days after the application of AN, at 228 ng/g-ww, and in the stems and leaves 24 days after the application of AN, at 39.5 ng/g-ww and 55.9 ng/g-ww, respectively.

4. Discussion

4.1. Relationship between Anthracene Concentration in Soil and Concentration in Radishes

Figure 4 shows the relationship between the AN concentration in the soil and the concentration in radishes. In the figure, “●” indicates the common logarithmic value of the detected concentration in both soil and radishes in P1, P2, and P3, and “○” indicates the value in P4. While P1, P2, and P3 were used in an experiment to grow plants from sowing onward in AN-contaminated soil and P4 was used in a methodologically different experiment to grow plants after maturity in AN-contaminated soil, positive correlations were observed in each experiment. It is inferred that the greater the degree of soil contamination, the greater the effect will be on the amount of AN assimilated by the radishes.

Figure 5 shows the relationship between the number of days that elapsed after exposure to AN and Cr/Cs and the ratio of the concentration in radishes to the concentration in the soil. A higher Cr/Cs ratio indicates that the chemical is more likely to be absorbed or concentrated in living organisms from the soil. The relationship between the number of days that elapsed after the application of AN (x) and the concentration ratio (y) was expressed as y = 0.201e^−0.113x. The correlation coefficient was 0.856, and the concentration ratio decreased exponentially. It was interpreted that AN was significantly absorbed in the early stages of radish growth, and some of the AN returned to the soil as the growth period progressed. For diazinon, the relationship y = 0.395e^−0.151x has been reported [28]. The Cr/Cs ratio for AN was lower than the ratio for diazinon. The lower Cr/Cs ratio for AN probably indicates that the lower solubility of AN makes movement between the radish plant and the soil difficult.

4.2. Concentration Ratio and Content Composition Ratio of Anthracene by Radish Part

Figure 6 shows the change over time in the concentration ratio of AN in radish organs in P4. The concentration ratio in the roots and stems was high at 1 day after AN application (15.1), but it declined sharply to 4.4 at 4 days after application. The concentration ratio subsequently increased to 18.8 at 24 days after AN application. The concentration ratio in the roots and leaves was 1.2 at 1 day after AN application and declined to 0.04 at 4 days after application; it subsequently increased (with fluctuations) to reach 13.2 at 24 days after application. The observed concentration ratios were higher than 1, except for those for the roots and leaves at 4 days after AN application. Root vegetables, such as radish, readily absorb polynuclear aromatic hydrocarbons from the soil, owing to their high penetration capacity [31,32,33]. It is likely that AN, with three-ring PAHs, enters the radish root through contaminated soil [34,35]. Contaminants in radishes show different behaviors depending on their properties, such as the octanol/water partition coefficient and metabolization [29,36,37,38,39]. AN was detected in all parts of the radish plant, i.e., the roots, stems, and leaves. AN is translocated within the plant, accumulates in each part, and reaches an equilibrium. It is presumed that the ratios of AN in the roots were higher because the root was directly in contact with the contaminated soil.

Figure 7 shows the change over time in the content composition ratio of AN by radish parts in planters P1 to P4. The median AN content ratio in the roots was 68.4%, with a range of 2.9–92.0%. The median AN content ratio in the stems was 3.3%, with a range of 0.3–35.8%. The median AN content ratio in the leaves was 26.8%, with a range of 4.5–96.3%. The median was highest in the roots, followed by the leaves and then the stems. No characteristic tendencies were observed in the fluctuations in the change over time for each content ratio. The moisture content of the radishes by part under dehydrating conditions of 105 °C and the organic matter content of radishes by part under combustion conditions of 650 °C, obtained in separate experiments, yielded average moisture content values of 88.1%, 93.2%, and 94.4% and average organic matter content values of 87.1%, 81.9%, and 79.6% for the roots, leaves, and stems, respectively. Taking into consideration that AN is a hydrophobic substance with a logKow value of 4.45 [40], as well as the water content and organic matter content of radishes by part, it is thought that AN in the soil is absorbed through the roots to circulate within the organism, and it is preferentially distributed and accumulated in the roots and leaves, which have high organic matter content.

5. Conclusions

The present study aimed at understanding the absorption and concentration of anthracene (AN) in the garden radish. Here, empirical data for AN absorption and accumulation in the vegetable radish (Raphanus sativus) were analyzed and their implications considered. Experiments were conducted to grow radishes from the sowing stage in soil contaminated with AN and to grow radishes in soil contaminated with AN following maturity (27 days after sowing). In both experimental conditions, the relationship between the AN concentration in the soil and those in the radishes were positive. The radishes absorbed greater AN quantity when the concentration in the soil was higher. The concentration ratio (Cr/Cs) decreased exponentially with the number of days following AN application. This suggests that AN is significantly absorbed in the early stages of growth, and it returns to the soil as the growth period progresses. The lower Cr/Cs for AN probably indicate that its lower solubility made movement between the radish and soil difficult compared to the result of a previous study. According to the time series of the part-specific AN concentration ratios and AN content, it seems that the AN in the soil was taken up through the roots to circulate within the organism, and it was preferentially distributed and accumulated in the roots and leaves, which had organic matter content.