[他誌掲載論文]
Journal of Pesticide Science, 2015, 40(4), 175～183 より転載

Relationship between plant uptake of pesticides andwater-extractable residue in Japanese soils
Yutaka Motoki1, Takashi Iwafune2, Nobuyasu Seike1,
Takashi Otani1 and Yoshihiro Akiyama2
1

National Institute for Agro-Environmental Sciences,

3–1–3 Kannondai, Tsukuba, Ibaraki 305–8604, Japan
2

Food and Agricultural Materials Inspection Center, Agricultural Chemicals Inspection Station,
2–772 Suzuki-cho, Kodaira, Tokyo 187–0011, Japan・本論文の版権は、日本農薬学会が所有していますが、版権所有者の転載許可を得て転載しています.

J. Pestic. Sci. 40(4), 175–183 (2015)
DOI: 10.1584/jpestics.D15-017

Original Article

Relationship between plant uptake of pesticides
and water-extractable residue in Japanese soils
Yutaka Motoki,1,* Takashi Iwafune,2 Nobuyasu Seike,1Takashi Otani1 and Yoshihiro Akiyama2
1
National Institute for Agro-Environmental Sciences,
3–1–3 Kannondai, Tsukuba, Ibaraki 305–8604, Japan
2
Food and Agricultural Materials Inspection Center, Agricultural Chemicals Inspection Station,
2–772 Suzuki-cho, Kodaira, Tokyo 187–0011, Japan(Received February 25, 2015; Accepted June 23, 2015)
The relationship between pesticide concentrations in Komatsuna (Brassica rapa var. perviridis) shoots and the extractable concentrations in soils by a sequential soil-extraction method was investigated in 8 pesticides and in 4 soils. Concentrations ofmany pesticides in Komatsuna shoots showed higher positive correlation with water-extractable than with total-extractable soil
concentrations. We also examined the effects of the soil-aging periods (SAPs) between pesticide applications and sowing on
pesticide concentrations in Komatsuna shoots. Pesticide concentrations in Komatsuna shoots and soil-water extracts decreased
with increasing SAPs. These results imply that estimating the pesticide concentrations in crops based on water-extractable concentrations in soil and setting the appropriate SAPs prevent contamination of crops by pesticide residues in soil. However, thecorrelation between pesticide concentrations in Komatsuna shoots and water extracts in soils, and the effectiveness of pesticide
reduction in Komatsuna shoots by setting long SAPs differed according to the pesticide type. Hence, the applicability of thewater-extraction method should be verified for each pesticide. © Pesticide Science Society of Japan
Keywords: pesticides, plant uptake, succeeding crops, water-extraction of soil, phytoavailability, soil aging.
Electronic supplementary materials: The online version of this article contains supplementary materials (Supplemental Figures
S1–S7, Supplemental Tables S1–S11), which are available at http://www.jstage.jst.go.jp/browse/jpestics/.

Recent studies of crops contaminated by persistent organic
pollutants (POPs), including dieldrin and heptachlor exo-epoxide, showed that application of carbonaceous adsorbents, suchas activated carbon, to soils4–6) and a grafting technique using a
low-uptake rootstock4,7) effectively decreased concentrations ofPOPs in cucurbit crops. However, the pesticides on the market
are more degradable than POPs in soils. Therefore, estimating
the residue concentrations in crops based on those in soils, followed by setting appropriate PBIs, is considered as one of realistic measures for preventing the production and circulation ofcontaminated crops because there is no cost for the materials.
Understanding the dissipation behavior of pesticides in soils andthe relationship between the residual levels of pesticides in soils
and those in crops is necessary for setting appropriate PBIs.
Residual levels of pesticides in soils are generally determinedby using a vigorous extraction technique of soils with an organic
solvent, such as acetone. However, assuming that some pesticides sorbed to soil particles are eluted into soil water and, subsequently, taken up by plant roots, a vigorous extraction technique that extracts not only water-extractable pesticides but alsosoil-sorbed pesticides is inadequate for assessing the amountof phytoavailable pesticides in soils. In fact, Sugimura et al.8,9)

Introduction
In Japan, some agricultural chemicals were recently detected incrops that exceeded the uniform residue limit of 0.01 ppm set
by the Japanese Positive List System.1) One reason for this is thatsucceeding crops take up from the soil residual pesticides applied to previous crops. Under the Japanese system of registering newly developed pesticides, the studies for pesticide residue
in succeeding crops are required only if the half-lives of pesticides in the soil are longer than 100 days2); therefore, in manycases, residue studies for succeeding crops are not conducted. In
contrast, in the United States, confined accumulation studies forrotational crops are required as a first tier evaluation for all pesticides except for those applied only on semi-permanent or permanent crops3); these results are available for setting appropriate
plant-back intervals (PBIs), i.e., plant restriction intervals, which
are included on the pesticide label.
* To whom correspondence should be addressed.
E-mail: ymotoki@affrc.go.jp
Published online October 28, 2015
© Pesticide Science Society of Japan

50

176 Y. Motoki et al.

Journal of Pesticide Science

concentrations in Komatsuna.

found that the phytotoxicity of pendimethalin and metolachlorin upland soils increased with increasing the concentration ofingredients extracted from soils by distilled water. Although
they did not provide the residual concentration of the ingredients in plants, their results imply that quantifying the amount ofwater-extractable pesticides in soils is important for predicting
the amount of plant uptake from soils. The water extractability of pesticides in soils depends on soil sorption. It is generally
known that the main sorptive medium of nonionic pesticides insoil is organic matter, i.e., neutral organic chemicals, including
pesticides that sorb to soil organic matter via hydrophobic interactions.10) Furthermore, it has been reported that the sorptionof a given pesticide shows high variability among Japanese soils
because of andosol, the typical upland soil in Japan, which contains high organic carbon (OC) content.11) Therefore, the phytoavailability of pesticides in Japanese soils is also likely to differ
considerably.
The objectives of this study are to investigate the variability of pesticide concentrations in Komatsuna (Brassica rapa var.
perviridis), as a representative of crops, cultivated in four typical Japanese soils, to assess the relationships between pesticideconcentrations in Komatsuna and water-extractable concentrations in soils, and to elucidate the effect of the soil-aging periods(SAPs) between applications of pesticides and sowing on residue

Materials and Methods
1. Pesticides
The eight pesticides used in this study (chemical purity >97.5%)
were purchased from Wako Pure Chemical Industries, Ltd.
(Osaka, Japan), Kanto Chemicals (Tokyo, Japan), and Dr. Ehrenstorfer GmbH (Augsburg, Germany) (Table 1). The pesticides were chosen based on their hydrophobicity, i.e., the rangeof their octanol-water partition coefficients (log Kow). To enable
simultaneous analysis, the pesticides were divided into two analytical groups.
2. Plant uptake experiments
Four typical Japanese agricultural soils were selected to covera wide range of OC contents (Table 2). Soil samples were airdried, passed through a 2.0-mm sieve, and used in pot experiments. To evenly mix pesticides with soils, a mixture of Celite
powder and pesticides was prepared.17,18) Approximately 1.5 L ofacetone solution containing 80 mg of each pesticide was added
to 400 g of Celite 545 (Wako Pure Chemical Industries, Ltd.),
and the acetone was evaporated for 5 hr at room temperature ina draft chamber. The concentrations of pesticides in the Celite
powder were quantified by acetone extraction; they ranged from

Table 1. Octanol-water partition coefficient (log Kow) and analytical groups of test compounds
CompoundIUPAC nameSubstance grouplog Kowa)

log Kowb)

log Kowc)

Analytical groupd)

Imidacloprid(E)-1-(6-chloro-3-pyridylmethyl-N-nitroimidazolidin-2-ylideneamine

Neonicotinoid

0.57

0.57

0.57

A

Clothianidin(E)-1-(2-chloro-1,3-thiazol-5-ylmethyl)-3-methyl2-nitroguanidine

Neonicotinoid

0.91

0.70

0.70

A

Thiacloprid(Z)-3-(6-chloro-3-pyridylmethyl)-1,3-thiazolidin2-ylidenecyanamide

Neonicotinoid

1.26

0.74

1.26

A

Metalaxyl

Methyl N-(methoxyacetyl)-N-(2,6-xylyl)-dl-alaninate

Phenylamide

1.65

1.75

1.75

A

Fosthiazate(RS)-S-sec-butyl O-ethyl 2-oxo-1,3-thiazolidin3-ylphosphonothioate

Organophosphate

1.68

1.68

1.68

A

Flutolanil

α,α,α-trifluoro-3′-isopropoxy-o-toluanilide

Phenylbenzamide

3.17

3.17

3.77

B

Procymidone

N-(3,5-dichlorophenyl)-1,2-dimethylcyclopropane-1,2-dicarboximide

Dicarboximide

3.30

3.14

3.30

B

Tetraconazole(RS)-2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol-1-yl)
propyl 1,1,2,2-tetrafluoroethyl ether

Triazole

3.56

3.56

3.53

B

a)
log Kow values were obtained from the Footprint Pesticide Properties Database of IUPAC.12) b) log Kow values were obtained from The Pesticide Manual(16th ed.).13) c) log Kow values were obtained from The 2011 Pesticide Handbook.14) d) Analytical methods are described in Supplemental Figs. S1–S3.

Table 2. Properties of test soilsa)
Soil

a)

Classificationb)

Texturec)OCd) (%)

CECe) (cmol(+)/kg)

Clay (%)

pH (H2O)

ECf) (mS/cm)

PS1

Yellow soil

Light clay

1.02

11.4

39.0

5.3

0.09

PS2

Gray lowland soil

Silty clay

1.46

18.2

25.3

5.8

0.17

PS3

Andosol

Loam

5.21

33.8

10.8

5.5

0.15

PS4

Andosol

Silty loam

8.65

35.4

1.8

5.8

0.05

11)

Data were obtained from our previous report. Soil samples PS1, PS2, PS3, and P4 correspond to Soils S3, S5, S7, and S8 in the previous report, respectively. b) According to the criteria adopted by the Cultivated Soil Classification Committee.15) c) According to the standards developed by the International Society of Soil Science.16) d) Organic carbon content.e) Cation exchange capacity. f) Electrical conductivity.

51Vol. 40, No. 4, 175–183 (2015)

Plant uptake of water-extractable pesticides in soils 177

84 to 137 µg/g. Each air-dried soil sample (PS1, 630 g; PS2, 550 g;
PS3, 450 g; PS4, 450 g), in the amount one hundred times greater than the amount of the Celite powder, was spread in a plastic bag; then Celite powder, 2 g of chemical fertilizer [0.16 g of
N as (NH4)2SO4, 0.07 g of P as Ca(H2PO4)2 ·H2O, and 0.14 g of
K as KCl], and 2 g of dolomite were evenly sprinkled onto thesoils and mixed well. The theoretical concentrations (µg/g) of
pesticides in air-dried soils were as follows: imidacloprid, 1.31;
clothianidin, 1.33; thiacloprid, 1.37; metalaxyl, 1.24; fosthiazate,
1.33; flutolanil, 1.06; procymidone, 0.84; tetraconazole, 1.10.
Plastic pots (600 mL) were filled with the soil samples. To
improve the germination rate, approximately 5 g of perlite wassprinkled onto the soil surface and lightly mixed. Each pot was
set in a plastic tray and placed in a climate chamber. The seedsof Komatsuna (B. rapa var. perviridis ‘Yokattana’) were sowedin each pot after 7 days for soils PS1 and PS4 and after 7, 35,
and 63 days for soils PS2 and PS3. Thus, an SAP of 7 days was
set for all soils as a base, and SAPs of 35 and 63 days were additionally set for soils PS2 (gray lowland soil) and PS3 (andosol).
Komatsuna was chosen because it is a popular leafy vegetable inJapan, is easy to cultivate, and grows rapidly. The water content
of the soils in the pots was adjusted by bottom-up irrigation to
between approximately 50% and 70% of their water holding capacity (WHC), except for on sowing day (90% of WHC). The
climate chamber was set to a cycle of 13 hr of light and 11 hr ofdarkness at 25°C, except during the first 7 days after the application of Celite to the soil and the period between sowing and germination (24 hr of darkness at 25°C). During the later growing
period, i.e., the 18-day period until harvest, 50 mL of liquid fertilizer (at two-fold the strength of Otsuka Chemical A prescription [mg/L]: N, 520; P, 104; K, 672; Ca, 328; Mg, 72; Mn, 2.4; B,
1.0; Fe, 5.4; Cu, 0.06; Zn, 0.18; Mo, 0.06) was applied 10 times toeach pot by bottom-up irrigation. Twenty-eight days after sowing, the shoots, including the leaves and stem, were harvested.
The fresh weight (FW) of the shoots ranged from 59.7–87.8 g per
pot. Pot experiments were performed in quadruplicate.
The shoot samples were frozen and finely ground using a
Grindomix GM200 (Retsch GmbH, Haan, Germany). Tengrams of aliquot was soaked in 80 mL acetone for 30 min andhomogenized using a Polytron® PT3100 (Kinematica AG, Lucerne, Switzerland) for 5 min. The solution was filtered througha 0.8-µm glass fiber filter and the volume was made up to
100 mL with acetone.

quential extraction method.
4. Sequential extraction of soils
Water extraction of the soils was carried out in accordance withthe standard batch sorption test method,19) although distilledwater rather than a solution of calcium chloride was used as theaqueous solvent. Twenty-five milliliters of distilled water wasadded to approximately 5 g dry weight (DW) of soil in a 50-mL
glass centrifuge tube. The tubes were agitated on a thermostat
shaker (TAITEC, Saitama, Japan) for 24 hr at 25±2°C in thedark. After shaking, the mixtures were centrifuged at 1200×g
for 30 min. Fifteen milliliters of supernatant was taken and used
to quantify the water extracts. In addition, 30 mL of acetone wasadded to the remaining sample, the tubes were shaken in a thermostat shaker for 20 min at 25±2°C and centrifuged at 1200×g
for 10 min, and the supernatant was carefully decanted. This extraction procedure was repeated twice. The collected supernatant was evaporated in a rotary evaporator to reduce the volume
to approximately 10 mL and then used to quantify the acetone
extracts.
The mass fraction of water-extractable pesticides in the soil,
Cwater (µg/g DW), was calculated using the following equation:

Cwater = Caq ⋅ (Vadd +Vsw ) / M ,(1)

where Caq (µg/mL) is the mass concentration of pesticide in
the aqueous phase after shaking for 24 hr, Vadd is the volume ofadded distilled water (mL), Vsw is the volume of soil water (mL),and M is the soil’s dry mass (g). The mass fraction of total extractable pesticides in the soil, Ctotal (µg/g DW), was represented
by the following equation:

Ctotal = (Caq ⋅ Valiq + mE ) / M ,(2)

where Valiq is the volume of the aliquot taken from the supernatant (mL) and mE is the mass of the pesticides extracted byacetone from the remaining sample after Valiq was removed (µg).
The half-lives (τ1/2) of Cwater and Ctotal were calculated using
the following equations18,20):

k = − [ln(Ct / C0 )]/ t(3)

τ1/2 = − ln(2) / k ,(4)where k is the first-order rate constant (1/day), Ct is the Cwater or
Ctotal after t days, and C0 is the initial Cwater or Ctotal.
The mean Cwater or Ctotal values during the periods from t1 to
t2 were calculated by integrating equation (3) from t1 to t2 anddividing the integration values by the periods (t1 to t2).18)

3. Soil dissipation experiments
Soil dissipation experiments were performed in triplicate under
the same conditions as the plant uptake experiments except thatplanting of Komatsuna and application of perlite and additionalliquid fertilizer were not carried out. Approximately 20 g of soil
was taken from each pot at 7, 14, 21, and 35 days for soils PS1and PS4, and at 7, 14, 21, 35, 63, and 91 days for soils PS2 andPS3 after Celite was applied to the soil. Each soil sample wasdivided into two subsamples: one was dried at 110°C to measure
the moisture content, and the other was analyzed using a se-

5. Pesticide analysis
Acetone extracts from Komatsuna shoots were analyzed toquantify the pesticides using two different methods, as shownin Supplemental Figs. S1 and S2. For group A, 10 mL of aliquot
was taken from each acetone extract and evaporated in a rotary
evaporator; 10 mL of acetonitrile was then added to each concentrate. The mixtures were cleaned with an ENVI-Carb II/PSA

52

178 Y. Motoki et al.

Journal of Pesticide Science

6. Statistical analysis
All statistical analyses, including linear regression analysis andanalysis of variance (ANOVA), were performed using SPSS Statistics 19 (IBM Corporation, Armonk, USA).

column (500 mg/500 mg; Supelco, Bellefonte, USA). For group
B, a 50-mL aliquot of each acetone extract was evaporated andcleaned with a diatomite column (InertSep K-solute 20 mL; GL
Sciences, Tokyo, Japan) followed by an ENVI-Carb II/PSA column (500 mg/500 mg; Supelco).
The aliquots of water extracts and concentrates of acetone extracts from soils were also analyzed using two different methods,11) whereas group B was analyzed using the method applied
for the analysis of Komatsuna shoots (Supplemental Fig. S2).
The samples from group A were cleaned with a diatomite column (InertSep K-solute 20 mL; GL Sciences) followed by a PSA
column (500 mg; Supelco) (Supplemental Fig. S3).
Cleaned samples were analyzed by liquid chromatographytandem mass spectrometry (LC-MS/MS) for group A (Supplemental Tables S1 and S2) and gas chromatography-mass
spectrometry (GC-MS) for group B (Supplemental Tables S3and S4). In the case of the high matrix effects of purified samples, matrix-matched standards were used in LC-MS/MS and
GC-MS.21)
Pesticide recovery tests were performed on pulverized samples of Komatsuna shoots, distilled water, and test soils. Komatsuna shoots (10 g) and distilled water (15 mL) spiked at 3 and1 ng/g, respectively, for all groups of pesticides. However, eachsoil (5 g) spiked at 5 and 3 ng/g for group A and group B pesticides, respectively. Those samples were analyzed using theabove-mentioned methods; however, 10 mL of distilled water
was added to soil samples prior to acetone extraction. The mean
recovery from five replicates for all compounds, except distilled water and soils for group A (four replicates), ranged from
73.3–113.0%; the coefficients of variation (CVs) were below
14.4% (Supplemental Tables S5–S7). The limits of quantification (LOQs) for pesticide analysis were calculated in accordancewith Japanese Industrial Standard (JIS) K 0312.22) The LOQs
for all compounds in Komatsuna shoots, distilled water, and all
soil samples ranged from 0.94–2.09 ng/g, 0.16–1.29 ng/mL, and
0.57–3.16 ng/g, respectively (Supplemental Tables S5–S7).

Results and Discussion
1. Relationships between pesticide concentrations in Komatsuna
shoots and water- or total-extractable concentrations in soils
Fig. 1 shows the pesticide concentrations in Komatsuna shoots
cultivated in four soils with a 7-day SAP. The residue concentrations in Komatsuna shoots for a given pesticide varied among
test soils, and CVs of the concentrations for a given pesticide
ranged from 17.6% for metalaxyl to 123.5% for thiacloprid.
Pesticide concentrations in Komatsuna shoots given the residue concentration in soils were analyzed using linear regression
analysis between pesticide concentrations in Komatsuna shoots
cultivated in soils with a 7-day SAP and the mean Cwater or Ctotal
values during the 28-day period until harvest, i.e., the period between sowing and harvest, in four soils (Table 3 and Supplemental Figs. S4 and S5). The regression lines were calculated using
data containing the point (0, 0) because if the concentration insoil is zero, the concentration in Komatsuna shoot is also zero.
Although the coefficient of determination (R2) values differedaccording to the type of pesticide, the concentrations of pesticides, except for metalaxyl and fosthiazate, in Komatsuna shoots
were more strongly correlated with the Cwater than with the Ctotal.
This result suggests that pesticide concentrations in Komatsuna shoots were affected more by the Cwater than by the Ctotal,
which implies that the water-extraction method of soil is moresuitable than the total-extraction method for estimating residual
concentrations in crops. On the other hand, for metalaxyl andfosthiazate, the R2 values were also high when using the Ctotal.
The variability of metalaxyl and fosthiazate concentrations in
Komatsuna shoots was especially low; the CVs of these pesticides were less than 30%. Therefore, for these two pesticides,
the regression lines were calculated by a two-point data group,
the point (0, 0) and the others (Supplemental Fig. S5). Focusing on imidacloprid, clothianidin, flutolanil, and tetraconazole,Fig. 1. Pesticide concentrations in Komatsuna shoots grown in soils with a 7-day soil-aging period. Columns with the same letter are not significantly
different at p<0.05, according to ANOVA-protected Tukey’s multiple range testing. Error bars indicate standard deviations (n=4).

53

Vol. 40, No. 4, 175–183 (2015)

Plant uptake of water-extractable pesticides in soils 179

Table 3. Linear regression analysisa) between pesticide concentrations in Komatsuna shoots cultivated on soils with a 7-day SAPb) and extractable concentrations in four soils
R2
CompoundWater-extracts

No. soils
Mean conc. during
28-day until harvestc)

Conc. at sowing

Total-extracts
Conc. at harvest

Mean conc. during
28-day until harvestc)

Conc. at sowing

Conc. at harvest

Imidacloprid

4

0.733

0.740

0.662

0.332

0.330

0.412

Clothianidin

40.918*

0.936**

0.868*

0.462

0.499

0.490

Thiacloprid

40.975**

0.103

0.994**

0.123

0.170

0.062

Metalaxyl

4

0.648

0.787*

0.670

0.844*

0.961**

0.852

Fosthiazate

4

0.466

0.681

0.448

0.732

0.891*

0.750

Flutolanil

4

0.566

0.691

0.481

0.543

0.511

0.511

Procymidone

4

0.714

0.492

0.918*

0.489

0.551

0.359

Tetraconazole

4

0.976**

0.985**

0.967**

0.334

0.328

0.289

* Significant at p<0.05; ** significant at p<0.01. Regression lines were calculated using data containing the point (0, 0), i.e., n=5. Soil-aging period(SAP) between application of pesticides and sowing. c) Mean values during the period between sowing and harvest.
a)b)

the mean Ctotal of four test soils showed almost the same values,
whereas the mean Cwater values of two andosols (soils PS3 andPS4), which had relatively high OC content, tended to be lower
than those of non-andosoils (Supplemental. Figs. S4 and S5).
Therefore, one reason for the high variability of residue concentrations in Komatsuna shoots for a given pesticide is considered
to be the difference of the sorption strength, depending on the
OC content of each test soil. However, we must consider thatsoil sorption is influenced not only by the OC content but also
by the OC quality. For example, the sorption of PS4, which had
the highest OC content, was lower than that of PS3, due to thedifference in the chemical composition of the OC.11)
To prevent the production and distribution of contaminatedcrops, pesticide concentrations in crops should be estimatedbefore harvest with the appropriate technique. Table 3 shows acomparison of the R2 values between pesticide concentrations in
Komatsuna shoots and the mean Cwater values during the 28-day
period until harvest and the Cwater values at sowing or at harvest. The R2 for many pesticides showed almost the same values, even when any Cwater value was used. Hence, it is possible
to estimate the residue concentrations in Komatsuna shoots bymeasuring the Cwater at sowing. However, thiacloprid and procymidone showed the lowest R2 values when using the Cwater values
at sowing and the highest values when using the Cwater values
at harvest. Such results might be explained by the difference inthe dissipation of the Cwater of thiacloprid and procymidone during plant growth among test soils (Supplemental Tables S8–S11).
The τ1/2 values in PS1, PS2, PS3, and PS4 were 4, 20, 40, and 11
days for thiacloprid, and 13, 52, 56, and 23 days for procymidone, respectively. The τ1/2 values differed depending on the soil
type; τ1/2 was especially low in PS1 as compared with other soils.
Among the test soils, the Cwater was highest in PS1 at sowing,
indicating that the absolute decrease of Cwater in PS1 during plant
growth was considerably higher than that in other soils. Thus,
if a pesticide exhibits distinctly different dissipation of Cwateramong soils during plant growth, the estimation of the residue

Fig. 2. Linear regression analysis between pesticide concentrations in
Komatsuna shoots cultivated in four soils with a 7-day soil-aging period
and the mean concentrations of water extracts in soils during a 28-day period until harvest, i.e., the period between sowing and harvest. The regression line was calculated using data containing the point (0, 0), i.e., n=33.

concentrations in Komatsuna shoots using the Cwater values atsowing is inadequate, and prediction of the Cwater values at harvest is required. To predict the Cwater values at harvest, it is necessary to evaluate the dissipation behavior of Cwater. Many studies
have investigated dissipation of Ctotal, including newly developed
pesticides, whereas very few data for Cwater are available. The dissipation rate of Ctotal is different from Cwater, due to the longer τ1/2
of Ctotal as compared to that of Cwater as a whole (Supplemental
Tables S8–S11). Therefore, estimating pesticide residue in crops
using predicted values of Cwater at harvest requires further studyof the dissipation behavior of Cwater with respect to target pesticides and field soils in which crops are planted.
The applicability of the water-extraction method using Cwater
values at sowing differed with the type of pesticide; in somecases, to obtain more accurate levels of pesticides in Komatsuna
shoots, the prediction of Cwater values at harvesting period was
required. Therefore, the applicability of the method should beverified for each target pesticide based on the actual calibration

54

180 Y. Motoki et al.

Journal of Pesticide ScienceFig. 3. Comparison of the bioconcentration factor (BCFwater), which was calculated by dividing the concentrations in Komatsuna shoots by the mean
concentrations of water extracts in soil during a 28-day period until harvest, i.e., the period between sowing and harvest. Columns with the same letter are
not significantly different at p<0.05, according to ANOVA-protected Tukey’s multiple range testing. Error bars indicate standard deviations (n=4).

and 5–50 hr, respectively. In addition, the TSCF values of Briggs
et al.23) were corrected for degradation of chemicals in plantshoots. On the other hand, the present experiment was carriedout using soil during the 28-day period and the BCFwater values
were not corrected for plant metabolism. Therefore, there is apossibility that the experimental conditions, such as the exposure period, the presence or absence of soil, and correction forthe degradation of chemicals in a plant, affect the difference between the BCFwater and the TSCF previously reported. However,there are no available data on the metabolism of tested pesticides in Komatsuna, which are taken up by the roots. The reasons for the variability in BCFwater remain unexplained, warranting additional study. Therefore, at present, the water-extraction
method for estimating the amount of pesticide residue in crops
should not be applied across pesticides but only for a given pesticide.

curve.
On the other hand, when the data for all pesticides wereplotted simultaneously, the correlation between the concentrations in Komatsuna shoots and the mean Cwater values during
the 28-day period until harvest was poor (Fig. 2). Such a weakcorrelation may be caused by the difference in the translocation
property of pesticides to shoots. Hence, the bioconcentration
factor (BCFwater) was calculated by dividing the concentrations
in Komatsuna shoots by the mean Cwater during the 28-day period until harvest (Fig. 3).
The mean BCFwater value for a given pesticide varied between
0.03 and 2.14; three pesticides—metalaxyl, fosthiazate, andflutolanil—showed low BCFwater values. In general, it has been
postulated that translocation of organic chemicals to shoots is
influenced by hydrophobicity. Briggs et al.23) found that the relationship between the transpiration stream concentration factor(TSCF), defined as the concentration of chemicals in the transpiration stream divided by the concentration in the external
solution (i.e., hydroponic medium), and the log Kow could be
represented by a bell-shaped curve with a maximum TSCF ata log Kow of 1.78, while Dettenmaier et al.24) reported a negative sigmoidal relationship between the TSCF and the log Kow. In
the present study, however, the relationship between the BCFwater and the log Kow was quite different from either of the above.
The BCFwater values did not take the transpiration volume into
account. However, the transpiration volume could be ignored
when the pesticides were compared because the tested pesticides
were simultaneously applied to a pot. Therefore, the translocation property of pesticides to shoots was not the direct cause of
the difference in the BCFwater. On the other hand, the concentration of pesticides in plants is also affected by the plants’ metabolism. For example, Fujisawa et al.25) improved the plant uptake
models of nonionized pesticides using a metabolic parameter,
i.e., the degradation rate constant in plants. Briggs et al.23) and
Dettenmaier et al.24) studied the plant uptake of pesticides in hydroponic culture, and their experimental periods were 24–48 hr

2. Influence of soil-aging periods (SAPs) on pesticide concentrations in Komatsuna shoots
Fig. 4 shows the relationships between pesticide concentrations
in Komatsuna shoots and three different SAPs—7-day, 35-day,and 63-day—for PS2 and PS3. The residue concentrations in
Komatsuna shoots decreased with increasing SAPs. The ratiosof the residue concentrations in Komatsuna shoots cultivatedin soils with a 63-day SAP as compared to those cultivated insoils with a 7-day SAP were 29–90% (mean=50%) and 12–69%(mean=51%) in PS2 and PS3, respectively. Thus, the ratios differed according to the type of soil and the pesticide. Focusing
on the effect of the aging period on the residue concentrations
in soils, the ratios of the Ctotal values at sowing with a 63-day
SAP as compared to those at sowing with a 7-day SAP were
27–108% (mean=80%) and 37–119% (mean=90%) in PS2 andPS3, respectively. On the other hand, the ratios of the Cwater values at sowing with a 63-day SAP as compared to those at sowing with a 7-day SAP were 12–84% (mean=52%) and 17–69%(mean=43%) in PS2 and PS3, respectively. As mentioned above,55

Vol. 40, No. 4, 175–183 (2015)

Plant uptake of water-extractable pesticides in soils 181

Fig. 4. Effect of soil-aging periods (SAPs) on residue concentrations of pesticides in Komatsuna shoots cultivated in soils PS2 (A) and PS3 (B). Columns
with the same letter are not significantly different at p<0.05, according to ANOVA-protected Tukey’s multiple range testing. Error bars indicate standard
deviations (n=4).
Table 4. Linear regression analysisa) between pesticide concentrations in Komatsuna shoots cultivated in soils with three different SAPsb) and extractable concentrations in soils PS2 and PS3
R2
CompoundWater-extracts

No. soils
Mean conc. during
28-day until harvestc)

Conc. at sowing

Total-extracts
Conc. at harvest

Mean conc. during
28-day until harvestc)

Conc. at sowing

Conc. at harvest

Imidacloprid

2

0.922**

0.938**

0.882**

0.172

0.151

0.212

Clothianidin

20.781**

0.802**

0.736*

0.227

0.150

0.285

Thiacloprid

20.963**

0.908**

0.988**

0.003

0.045

0.004

Metalaxyl

2

0.149

0.239

0.022

0.686*

0.585*

0.545

Fosthiazate

2

0.339

0.476

0.204

0.787**

0.799**

0.756*

Flutolanil

20.936**

0.807**

0.988**

0.306

0.293

0.260

Procymidone

20.964**

0.868**

0.956**

0.207

0.270

0.120

Tetraconazole

2

0.931**

0.726*

0.977**

0.379

0.371

0.343

* Significant at p<0.05; ** significant at p<0.01. a) Regression lines were calculated using data containing the point (0, 0), i.e., n=7. b) Soil-aging period(SAP) between application of pesticides and sowing. SAPs were set to 7 days, 35 days, and 63 days. c) Mean values during the period between sowing andharvest.

the dissipation of Ctotal was slower than that of Cwater as a whole(Supplemental Tables S8–S11). Therefore, the reduction of Cwaterin soils caused by soil aging was larger than that of Ctotal. Several
studies reported that soil sorption of pesticides such as imidacloprid,26,27) nicosulfuron,28) and simazine29) increased during
the aging period; thus, it is implied that the dissipation of Cwater
is affected not only by general dissipation factors such as biodeg-

radation, hydrolysis, and volatilization, but also by increasing
soil sorption with increased aging time.
To verify the effectiveness of pesticide reduction in Komatsuna shoots by setting long SAPs for a given residue concentration
in soils, we elucidated the correlation between pesticide concentrations in Komatsuna shoots cultivated in soils with three different SAPs and the extractable residue in PS2 and PS3 (Table 456

182 Y. Motoki et al.

Journal of Pesticide Scienceand Supplemental Figs. S6 and S7). Similar to the four test soils
with a 7-day SAP (Table 3), the R2 values of the regression lines
calculated using Cwater were higher than those using Ctotal, except
for metalaxyl and fosthiazate. These results suggest that the water-extraction method of soil is more suitable for estimating pesticide concentrations in Komatsuna shoots cultivated in different soils with different SAPs than the total-extraction method.
However, the R2 values of the regression lines and the effectiveness of pesticide reduction in Komatsuna shoots by setting long
SAPs changed depending on the combination of pesticides andsoils. To apply the water-extraction method to target pesticides,
it is necessary to prepare actual calibration curves, i.e., regression lines, by using a target crop and field soils and to verify theapplicability of the method based on the calibration curve.
As discussed above, the dissipation of Cwater is considered toaffect the pesticide concentrations in Komatsuna shoots. In thepresent study, dissipation studies of pesticides in soils were performed with pot experiments, in which the water content of soils
was adjusted by bottom-up irrigation; thus, the dissipation ofpesticides in the pots appeared slower than that in fields withleaching of pesticides after rainfall or irrigation.30) Therefore,
the effectiveness of pesticide reduction in crops cultivated inpots by setting long SAPs may also be lower than that in fields.
Hashimoto et al.4) reported that application of activated carbon
to fields reduced dieldrin concentrations in cucumber fruits to
42–64% of those cultivated in non-treated fields. The effectiveness of dieldrin reduction in cucumber fruits by applying activated carbon roughly corresponds to the results obtained in thepresent study by setting long SAPs. Therefore, although the effectiveness of pesticide reduction differs depending on the typeof pesticide and the soil, for pesticides that are available on themarket, which show faster dissipation in soils than POPs, setting
appropriate SAPs is suggested as one of practical measures toreduce pesticide concentrations in crops.

Acknowledgements
The authors thank Prof. H. Watanabe of Tokyo University of Agriculture
and Technology for his helpful suggestions. The test soils were kindly supplied by Aichi Agricultural Research Center and Tochigi Prefectural Agricultural Experiment Station. This research was supported by the Environmental Research and Technology Development Fund (5-1302) of the
Ministry of the Environment, Japan.

References
1) S. Hori: Foods Food Ingredients J. Jpn. 212, 496–508 (2007) (in Japanese with English summary).
2) Agricultural Materials Division, Agricultural Production Bureau,
Ministry of Agriculture, Forestry and Fisheries of Japan: The application of “The guidelines related to the study reports for the registration application of pesticide,” Notification No. 13-Seisan-3986, Oct10, 2001 (Final amendments were made on May 15, 2014) (in Japanese), http://www.acis.famic.go.jp/shinsei/3986/3986.pdf (Accessed
27 July, 2015).
3) U.S. EPA: Residue Chemistry Test Guidelines “OPPTS 860.1850 Confined Accumulation in Rotational Crops,” Aug. 1996.
4) Y. Hashimoto: J. Pestic. Sci. 32, 229–234 (2007).
5) H. Murano, T. Otani, T. Makino, N. Seike and M. Sakai: Soil Sci. Plant
Nutr. 55, 325–332 (2009).
6) T. Saito, T. Otani, N. Seike, H. Murano and M. Okazaki: Soil Sci.
Plant Nutr. 57, 157–166 (2011).
7) T. Otani and N. Seike: J. Pestic. Sci. 32, 235–242 (2007).
8) H. Sugiyama, K. Komamiya and K. Kobayashi: Weed Res. Jpn. 35,
122–128 (1990) (in Japanese with English summary).
9) H. Sugiyama and K. Kobayashi: Weed Res. Jpn. 38, 300–306 (1993).
10) R. D. Wauchope, S. Yeh, J. B. Linders, R. Kloskowski, K. Tanaka, B.
Rubin, A. Katayama, W. Kördel, Z. Gerstl, M. Lane and J. B. Unsworth: Pest Manag. Sci. 58, 419–445 (2002).
11) Y. Motoki, T. Iwafune, N. Seike, T. Otani and M. Asano: J. Pestic. Sci.
39, 105–114 (2014).
12) http://sitem.herts.ac.uk/aeru/iupac/ (Accessed 26 Nov., 2014).
13) C. MacBean (ed.): “The Pesticide Manual,” 16th Ed., British Crop
Protection Council, Surrey, 2012.
14) The 2011 Pesticide Handbook Editorial Committee: “The 2011 Pesticide Handbook,” Japan Plant Protection Association, Tokyo, 2011 (inJapanese).
15) Cultivated Soil Classification Committee: Misc. Publ. Natl. Inst. AgroEnviron. Sci. 17, 31–55 (1995) (in Japanese).
16) International Society of Soil Science: Minutes of the First Commission Meetings, International Congress of Soil Science, Washington,
1927, Proc. Congr. Int. Soc. Soil Sci. 4, 215–220 (1929).
17) T. Otania, N. Seike and Y. Sakata: Soil Sci. Plant Nutr. 53, 86–94(2007).
18) S. Namiki, T. Otani and N. Seike: Soil Sci. Plant Nutr. 59, 669–679(2013).
19) OECD: “Guidelines for Testing of Chemicals,” Section 1 (106),
OECD, Paris, 2000.
20) OECD: “Guidelines for Testing of Chemicals,” Section 1 (111),
OECD, Paris, 2004.
21) J. Zrostlíková, J. Hajšlová, J. Poustka and P. Begany: J. Chromatogr. A
973, 13–26 (2002).
22) Japanese Standards Association: JIS K 0312, Tokyo, Japan (2005) (inJapanese).
23) G. G. Briggs, R. H. Bromilow and A. A. Evans: Pestic. Sci. 13, 495–

Conclusion
The water-extraction method of soils was more effective for estimating pesticide concentrations in crops cultivated in different soils with different SAPs than the total-extraction method.
Furthermore, pesticide concentrations in Komatsuna shoots
decreased by setting long SAPs. These results imply that estimating pesticide concentrations in crops based on the Cwater andsetting appropriate SAPs prevent production and distributionof crops contaminated with pesticide residues from soils. However, the applicability of the water-extraction method and theeffectiveness of pesticide reduction in crops by setting long SAPs
differed, depending on the combination of pesticides and soils.
Therefore, a calibration curve for a target pesticide should be
individually prepared by using the crop of interest and the fieldsoil in which the crop is planted; the applicability of the waterextraction method should be verified from the results. Furthermore, the dissipation studies of Cwater in actual fields are required
to estimate the effectiveness of setting long SAPs for the reduction of pesticides in crops.

57Vol. 40, No. 4, 175–183 (2015)

Plant uptake of water-extractable pesticides in soils 18327) M. Oi: J. Agric. Food Chem. 47, 327–332 (1999).
28) J. B. Regitano and W. C. Koskinen: J. Agric. Food Chem. 56, 5801–
5805 (2008).
29) J. B. Regitano, W. C. Koskinen and M. J. Sadowsky: J. Agric. Food
Chem. 54, 1373–1379 (2006).
30) K. Müller, G. N. Magesan and N. S. Bolan: Agric. Ecosyst. Environ.
120, 93–116 (2007).

504 (1982).
24) E. M. Dettenmaier, W. J. Doucette and B. Bugbee: Environ. Sci. Technol. 43, 324–329 (2009).
25) T. Fujisawa, K. Ichise, M. Fukushima, T. Katagi and Y. Takimoto: J.
Agric. Food Chem. 50, 532–537 (2002).
26) L. Cox, W. C. Koskinen and P. Y. Yen: Soil Sci. Soc. Am. J. 62, 342–347(1998).

58