THE RAPID ANALYSIS FOR DIOXIN DERIVED FROM AGENT ORANGE IN SOIL~Ⅱ ANALYTICAL METHOD WITH FLOW IMMUNOSENSOR ~Tateishi N1, Takagi Y1, Oshima S1, Ueda Y2, Watanabe I2, Binh MD3, Trung NQ3, Honda K2
1

Kyoto Electronics Manufacturing Co., Ltd., 68 Ninodan-cho, Shinden, Kisshoin, Minami-ku, Kyoto 601-8317,
Japan; 2Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, 790-8566, Japan; 3Vietnam
Academy Of Science And Technology, Institute Of Environmental Technology, A30 Bld., 18 Hoang Quoc VietStr., Hanoi.

Introduction
In Da Nang airport Middle Vietnam, decontamination of Agent Orange contaminated soil is started bycooperation projects the U.S. Agency for International Development (USAID) and the Ministry of Defence ofVietnam in 2012, Interest in soil survey has increased in Vietnam this opportunity. In Vietnam, the effective useof degraded land of about 9 million ha, spread the cause of soil contamination by Agent Orange during the war
and slash-and-burn is required. When the use of degraded land, the analysis of 2,3,7,8-TCDD(D48) which is abyproduct of Agent Orange is a problem1),2),3) for a wide range of soil in Vietnam, since the only use of
instrumental analysis that requires expensive equipment and advanced technology is not realistic, to adopt more
quick, simple, and inexpensive analytical method is desirable.
In recent years, more than 175 countries are working on reducing Persistent Organic Pollutants (POPs) thatseem to take immediate steps based on treaty established on the purpose of building the international framework.
Regular monitoring is required for the understanding of pollution by POPs in the environment in particular.
Technological development of bio-assay has made dizzying development because the need for simple, rapid,
inexpensive analytical technique is increasing in Japan.4).
By the contamination of substances that cannot assume in the environment, the accuracy of analysis is reducedin bioassays, to ensure the reliability is difficult. In order to solve these problems, we developed a trace PCBs
analysis system in insulating oil and dioxin analysis system in the exhaust gas by combining the automatic
pretreatment device which has a high refining capacity and flow immunosensor using antibodies were developed
strategically. And then, these systems were certified as an analytical device with high reliability5). It is to performthe antibody development and measurement system constructed corresponding as a particularly important pointin the development of this sensor. Since it is necessary to calculate toxicity equivalent amount (TEQ) the amountof dioxins, targeting indicators isomer highly contribute to the TEQ of dioxins in incineration samples ofinsulating oil in the Dioxin in exhaust gas it is to target a wide PCB isomer group having a common structure of
the major isomer of 3-6 chlorine contained in the product that had been using a PCB in the PCB analysis.
Similarly, in the development of the dioxin-contaminated soil analysis system derived from Agent Orange, it
should be noted that to be calculated TEQ dioxins unlike dioxin problem in the world is not the purpose.
Furthermore, the practical use of analytical techniques, for verification test using actual sample is essential that it
be examined by using a soil sample to be analyzed is also an important point of development.
Materials and Methods

1. Samples
The black soil and paddy soil (0.2pg/g or less HRGC/HRMS measured value of the D48) were used for blanktest. The standard soil sample (1000pg/g) which made by mixing paddy soil without dioxin and Celite (KantoChemical) that was added D48 hexane standard solution to be a 100ng/g in advance, was used for the test ofreproducibility confirmation. Simulated samples (100, 250, 500, 1000pg/g) were prepared by the method in
similar to standard soil samples were used for the test of accuracy confirmation. the standard soil of were used.
As real samples, 10 samples (2–5000pg-TEQ/g) were taken in Japan, 2 samples (about 1000pg-TEQ/g) weretaken in Vietnam was used.

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2. Reagents and instrument
Acetone and Hexane for pesticide residue and PCBs analysis weresupplied by Wako Pure Chemical Industries, Ltd. DMSO and Sulfuric
acid of special grade chemicals were supplied by Kanto Chemical Co.,
Inc. Hybridoma with D48 antibody was given from Food and Drug
Safety Center. This antibody reacts with the 2,3,7,8-TeCDD as shown inTable 1. Measurement cell and Buffer were supplied by Kyoto
Electronics Manufacturing Co., Ltd. A pretreatment device for Dxn
analysis ; SZ-DXN-PT010 (Seeds Tec Co., Ltd.) was used to prepare Fig.1 Flow Immunosensor DXS-610
soil samples. A flow immunosensor DXS-610 (Fig1, Kyoto Electronics
Manufacturing Co., Ltd.) on the principle of kinetic exclusion assay was used to measure. D48 hexane orDMSO solution which were exchanged from nonane (CIL's) one by solvent substitution was used as standardsolution.
3. Procedure of Sample Pretreatment
In the extraction step, collect soilsamples 5g first, and after the operationaccording to the flow shown in Fig.2,
2ml hexane crude extract was obtained
as the result.In the pretreatment step,
the separated 1.25g substantial amountof hexane crude extract ware applied topretreatment column, finaly DMSO
samples are obtained by heating
multilayer silica gel / alumina column
method shown in Fig.3 by using thesample preparation device6).

SystemSampling soil (air-dried)
5g soil in 50mL centrifugetube
The addition of 10mL of 4%0.1M
H2SO4 / Acetone

Ultrasonic Extraction

Sampling loading
1mL of n-Hexane ExtractHeating multi-layer column
60℃, 30min

Connecting concentration column

35-40kHz, 60min, 50℃

Centrifuge

n-Hexane elution

3500rpm, 5min

40mL, 15min

Acetone Extract
5mL extraction liquid in 40mL test tube
THe addition of 25mL of 5%NaCl-Waterand 2mL of n-Hexane

Extraction by the hand shaking
2min

Drying concentration column
85℃, 10min 10-15kPa

DMSO elution
85℃, 550μ L of DMSO, 5min

KinExA (DXS-610)

4. Immunosensing Methods of D48
n-Hexane Extract
A Flow Immunosensor DXS-610
Fig.3 Flow diagram of the pretreatment(Kyoto Electronics Manufacturing Co., Fig.2 Flow diagram of the extractionLtd.) for the principle of Kinetic
Exclusion Assay (Fig.4) was used to measure Dioxins. This cross reactivity of the antibody for 2,3,7,8-TeCDD
shown in Table 1. The detection limit of the measurement method shown in Fig.4.
The preparation of the measurement sample was stirred with 20μL DMSO in 80μL pretreated DMSO
samples, and was stirred after added 1.4ml sample preparation buffer. In addition to 0.5ml fluorescent labeledanti-D48 monoclonal antibodies (D48 Ab) prepared to the optimum concentration and gently stirred. 0.2ml
measurement sample solution was measured at a flow rate of 0.75mL/min by Flow Immunosensor.
Table 1. Cross Reactivity of D48 A.b.
Dioxin Isomer
2,3,7,8-TeCDD
1,2,3,7,8-PeCDD
1,2,3,4,7,8-HxCDD
1,2,3,6,7,8-HxCDD
1,2,3,7,8,9-HxCDD
1,2,3,4,6,7,8-HpCDD
OCDD
2,3,7,8-TeCDF
1,2,3,7,8-PeCDF
2,3,4,7,8-PeCDF
1,2,3,4,7,8-HxCDF
1,2,3,6,7,8-HxCDF
1,2,3,7,8,9-HxCDF
2,3,4,6,7,8-HxCDF
1,2,3,4,6,7,8-HpCDD
1,2,3,4,7,8,9-HpCDF
OCDF

Cross-reactivity (%)
Dioxin Isomer
Cross-reactivity (%)
100
3,4,4',5-TeCB<173,3',4,4'-TeCB
1<13,3',4,4',5-PeCB<1<13,3',4,4',5,5'-HxCB<1<12',3,4,4',5-PeCB<1<12,3',4,4',5-PeCB<1<12,3,3',4,4'-PeCB<192,3,4,4',5-PeCB<1<12,3',4,4',5,5'-HxCB<142,3,3',4,4',5-HxCB<1<12,3,3',4,4',5'-HxCB<1<1
2,3,3',4,4',5,5'-HpCB<1<1<1: means concentration of IC50 is 100<1times more than D48<1<1<1B/B0 1.01

Detection Limit: 0.04ng/mL

0.8
0.6
0.4
0.2

0.04
00.001

0.01

0.1

110

D48 conc. (ng/mL)
Fig.4 Comparison of Calibration Curve in D48 ofantibody.
We used D48 as standard substance

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Results and Discussion
1. The blank test and the spike recovery test
In the results of the blank test using the black soils and paddy soils, the measurement values ware 100pg/g orless, lower limit of quantitation of this measurement method.
The samples that was added a final concentration of 40pg/mL, and 80pg/mL to be cleaned-up black soils andpaddy soils were used for the recovery test. As the result of the recovery tests, each recovery rates were 100%(paddy soil) and 97% (black soils). And therefore we determined that there is no contamination of matrix
components affecting the measurement system in the soil samples which were prepared using the extraction andthe pretreatment method.
2. Reproducibility check from the extraction to the measurement
In Table 2, as to the standard soil samples the values andTable 2 Validation of Analysis system for D48.
the variation coefficient of the 3 times from extract process
to measured process (N=3) were showed. Coefficient of
Quantitative value
Average
Sample Times
pg/ g
pg/ g
variation is 4.6, the recovery rate was about 80 %. It is
confirmed that the present analysis is an analytical system
1790
A778
with high reprodusivility.
2738
3CV%
4.6806

3. Accuracy check for Flow Immunosensor (DXS-610)
The D48 values by DXS-610 and D48 and TEQ values by HRGC / HRMS was shown in Table 3.
The correlation plot of the D48 values by DXS-610 against those by HRGC / HRMS for 32 soil samples (16kinds) was shown in Fig.5. For all the samples, DXS-610
measurement results were obtained that is within the range of Table 3 Measurement result for soil samples
HRGC/ HRMS HRGC/ HRMS
DXS-61082-146% relative to the value of D48 HRGC/HRMS, further, theSample
Kind(pg-TEQ/ g)(pg-D48/ g)(pg-D48/ g)correlation diagram is good linearity through the origin.
No.1-1
91
91
N.D.
Therefore, this system was found to be an analytical method
No.1-2
91
91
N.D.
No.1-3
91
91
N.D.
which can be quantified accurately D48 in real soil samples.
Conclusion
This analysis system could also be applied to soil samples ofdifferent species and pollution from Japan and Vietnam, and wasable to accurately quantify the D48 in samples.
Therefore, it can not only take full advantage of themonitoring and screening of dioxin derived Agent Orange, it willcontribute to the production of dioxin pollution map in Vietnam.

No.2-1

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No.2-3No.3-1

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210
330

186
171
363

No.3-2
No.3-3

330
330

330
330

334
341

No.4-1
No.4-2

990
990

987
987

856
927

No.4-3No.5-1
No.5-2

990
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1659

987
1640
1640

811
1660
1704

1659
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1640
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1708
840

No.6-2
No.6-3

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803
787

No.7
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1.9
11
23

0.2
0.4
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N.D.
N.D.
N.D.

No.10
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40
97

1.6
4

N.D.
N.D.

150
330

714N.D.
13330
330
1200

141433141547

No.15-1
No.15-2

3100
3100

100
100

94
101

No.15-3

3100

100

103

No.16

5500

160

182

No.5-3No.6-1

Acknowledgment
This study was partly supported by Science and Technology
Research Partnership for Sustainable Development (SATREPS)
in JICA/JST for “Multi-beneficial measure for the mitigation ofclimate change in Vietnam and Indochina countries bydevelopment of biomass energy”.
References
1) Young, A, L.The History, Use, Disposition and Environmental
Fate of Agent Orange. Springer, New York, 2009
2) Mai Ta et al., Dioxin contaminationin soils of SouthernVietnam. Chemosphere. 2007,67(9),1802-1807
3) Masao Kishida et al., Chemosphere. 2010,78(2),127-133

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Pseudo-sample

Samplein Vietnam

Samplein Japan349

DXS-610 (pg-D48/g)4) Takagi Yoko et al., Persistent organic pollutants (POPs) research in Asia, 2008/ 10, 96–101.
5) Takashi Matsuki et al., Org Compounds 67, 39-41, (2005)
20006) Hong, J. et al., Chemosphere, 88, 1287-1291 (2012)
y = 1.00 x
R2 = 0.99

1500100050000500

1000

1500

2000

HRGC/HRMS (pg-D48/g)
Fig. 5 Correlation diagram(HRGC/HRMS value vs. DXS-610 value)●:Japan, ●: Vietnam

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