COMPARISON OF DIFFERENT CLEAN-UP, EXTRACTION AND DETECTIONTECHNIQUES FOR THE DETERMINATION OF DL-PCBs AND PCDD/Fs IN
SAMPLES FROM HIYOSHI CORPORATION IN JAPAN AND SOIL AROUND A
WASTE LANDFILL IN CHINA
Mao QJ1*, Lu SY1, Chen T1, Li XD1, Yan JH1, Cen KF1, Nakamura M2, Huang JQ212

State Key Laboratory of Clean Energy Utilization, Zhejiang University, Hangzhou, PR China;
Hiyoshi Ecological Services Co., Kitanoshyo 908, Omihachiman, Shiga 523-0806, JapanIntroduction
The Stockholm Convention came to be legally effective in China since Nov. 2004, therefore related emission
control and governance standards of persistent organic pollutants (POPs) should be legislated to implement theconvention plan in China1. However, general invest+igation on environmental background contamination
database is lack of, especially the survey on POPs contamination of soil is very limited in China. Sanitary
landfill is one of the main methods of waste disposal, but less attention has been paid to soil contamination in
landfill, compared with incinerator plants 2-4. Nine soil samples were collected surrounding the landfill, including
sites lying in the lower terrain of the landfill, soil covered on the waste and sites on the natural mountains aroundthe landfill. CALUX (Chemically Activated LUciferase gene eXpression) bioassay is considered to be a fast,sensitive and inexpensive tool for the analysis of a high number of samples 5, therefore the use of this technology
in routine analysis of soil samples may be a valuable alternative for HRGC/MS. Two kind of clean-up process
were operated in CALUX assay, i.e. manual and semi-auto, two dioxin-extraction methods were compared inCALUX assay, i.e. soxhlet and ultrasonic; as well as two dioxin analysis methods were compared in this work,
i.e. CALUX assay and HRGC/MS analysis. Polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinateddibenzofurans (PCDFs) and dioxin-like polychlorinated biphenyls (DL-PCBs) were analyzed in this work.
Materials and methods
The samples in this work were divided into two parts. The first six samples, including four soil and two fly ashsamples, which are of high concentration, were obtained from Hiyoshi Corporation in Japan. The left ninesamples, which are of respectively low concentration, were collected from a municipal solid waste landfill ineast China. The soil samples were collected by mixing five different aliquots (each in four main directions of 5mto the center) within a 25m2 area. The sampling was carried out by inserting a cylindrical steel corer down to a
20cm depth. Approximately 2 kg of soil was taken and mixed at each site. The soil samples were subsequently
dried in an electric thermostatic blast box under 100℃ within 3h. Then they were ground and passed through a2-mm sieve. About 500 g soil of each sample was homogenized through a 60-mesh sieve, and was stored inbrown vials away from light. Sources and numbers of nine soil samples are described in Table 1.
For Hiyoshi samples, two kind of clean-up process, i.e. conventional manual and semi-auto, were operated after
extraction for CALUX assay. Semi-auto process was operated in a new developed device which was under trial.
New-produced acid-silica gel and XCARB columns were used for clean-up instead of manual filled columns.
For the nine soil samples collected from the landfill in China, two dioxin-extraction methods, i.e. soxhlet andultrasonic, were applied in CALUX assay. Both CALUX assay and HRGC/MS were compared in analyzing
Hiyoshi samples and Chinese samples.
For Soxhlet Extraction (SxE), overall 7g (dry matter) of soil samples were taken out, and the SxE solution wasdivided into two parts: the first half was prepared for CALUX assay; the other half 3.5g of SxE samples wasrefrigerated before HRGC/MS analysis. For ultrasonic extraction (UsE), 3.5g of soil samples were dissolved in
10ml 20%methanol/toluene, and then the solution was shaken and ultrasonic extracted for 5min. The supernatant
after UsE was passed through a celite column, the above operations were conducted two times, and the mixed
solution was prepared for CALUX assay. The extractions both from SxE and UsE were further loaded onto an
acid-silica gel and XCARB (XDS Inc., USA) bigeminal columns for clean-up. The DL-PCBs and PCDD/Fs
fractions were then eluted with different solution only onto XCARB column respectively. The CALUX assay
was carried out using a recombinant H1L6.1 cell line. The cells were seeded on 96-well microplates, and after

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24h of incubation, cells were exposed in duplicate to the purified sample extracts and 2,3,7,8-TCDD standard
solution for 20-24h of incubation. Then luciferase activity was measured using an Ensipre luminometer (PerkinElmer). Duplicate ultrasonic sampling for CALUX assay has been done.
Four of the nine refrigerated 3.5g of SxE samples were taken out for HRGC/MS analysis. Clean-up andanalytical determinations were described as the previous research 6. Each sample was spiked with a mixture of13C-labelled PCDD/Fs compound stock solution (10µl, 100ng/ml) and clean-up standard (25µl, 8ng/ml) beforepickling. The extracts from SxE were subsequently passed through multilayer silica gel column and alkaline
alumina column following USEPA Method 1613. The extracts were blow down to 40µl by nitrogen (N2), and5µl internal standard solution (200ng/ml) were added before sample were subjected to PCDD/Fs analysis byusing HRGC/HRMS (JEOL JMS-800D) with a DB-5MS column (60m0.25mm0.25µm).
Results and discussion
The TEQ of DL-PCBs and PCDD/Fs processed by manual and semi-auto clean-up pretreatment are written as“CALUX-M” and “CALUX-A”, respectively. The results show that CALUX-M and CALUX-A of the six
samples from Hiyoshi Corporation are well correlated; the ratio between CALUX-M and CALUX-A is in therange of 0.8 to 1.2 (table not show in this paper). Taking the average of CALUX-M and CALUX-A as theCALUX-TEQ of the six samples, the TEQ of DL-PCBs and PCDD/Fs determined by HRGC/MS is written as“WHO-TEQ”. The ratio between CALUX-TEQ and WHO-TEQ were 1.8~2.9 for soil samples and 3.3~3.6 forfly ash (Fig. 1), so generally, CALUX-TEQ showed higher values than WHO-TEQ7.
TEQ values of the nine samples collected from a municipal solid waste landfill in China are listed in Table 2.
The TEQ of total DL-PCBs and PCDD/Fs determined by CALUX assay using ultrasonic and soxhlet extraction
are named as “CALUX-UsE” and “CALUX-SxE”, respectively. The TEQ of total DL-PCBs and PCDD/Fs
determined by HRGC/MS with WHO-TEF (2006) is named as “HRGC/MS”. Soil samples collected fromlow-lying sites (No. 1~2) and fresh soil covered on solid waste (No. 3~5) showed lower values than soil locatedon the mountains surrounding the landfill (No. 6~9). Average CALUX-UsE and CALUX-SxE values of samples
No. 1~5 are 1.2 pg-TEQ/g and 1.6 pg-TEQ/g, respectively; while for samples No. 6~9 are 21 pg-TEQ/g and 19
pg-TEQ/g respectively. The TEQ concentrations in all soil samples were at background level3, providing data
reference for the establishment of POPs control directory in China.
In comparison of soxhlet and ultrasonic extraction by CALUX assay, the ratio of UsE to SxE is centralized in therange of 0.6~1.2, the correlation coefficient is 0.956 (Fig. 2). These results show that both soxhlet and ultrasonicextraction meet the standard measurement in the pretreatment of soil in CALUX assay. The coefficient ofvariation (C.V.) values of the repeated UsE samples are below the required standard value of 30% except thefirst four samples with lower concentration below 1.8 pg-TEQ/g. Stability of the pretreatment of lower
concentration samples warrants further repeat.
CALUX-SxE and HRGC/MS of the four soil samples from landfill were 12~24 pg-TEQ/g and 9～17 pg-TEQ/g
respectively, these results show that CALUX and HRGC/MS are highly correlated in soil samples within low
concentration. The ratio between CALUX-TEQ and WHO-TEQ was 1.3  0.5, which is close to the result of1.4  0.7 in the previous research8.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (50706043), National NaturalScience Foundation of China (No. 51276162). This research work was financially supported by Hiyoshi
Corporation, Japan.
References
1. National Implementation Plan for the Stockholm Convention on Persistent Organic Pollutants, The People’s
Republic of China. (2007)
2. Ministry for the Environment. Dioxin concentration in residential soil, New Plymouth
3. Ott Roots, Bernhard Henkelmann, Karl-Werner Schramm. (2004); Chemosphere. 57: 337-342
4. Ministry of Environmental Protection of PR China, et al.. (2010) China Environ. Protech. Indust. 12: 11-13.
5. Du YZ, Lu SY, Yan JH, et al.. (2012);The 32nd International Symposium on Halogenated Persistent Organic
Pollutants and POPs. 74: 644-647
6. J.H. Yan, M.X. Xu, S.Y. Lu, X.D. Li, et al.. (2008); Journal of Hazardous Materials. 151: 522-530

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7. US Environmental Protection Agency, Test methods for evaluating solid waste, physical/chemical methods.
US EPA, 2007.
8. Du YZ, Yan JH, Chen T, Lu SY, et al.. (2010); The 30th International Symposium on Halogenated Persistent
Organic Pollutants and POPs. 72: 88-91
Table 1 Sources and numbers of the nine soil samples collected from a landfill in China
Sources of nine soil
Soil collected from
Fresh soil covered onSoil located on thesamples in China
low-lying sites of thewaste
mountains surrounding thelandfill
landfill
No. of soil samples
No.1, No.2
No.3, No.4, No.5
No.6, No.7, No.8, No.9
Table 2 TEQ Summary of the nine soil samples using UsE and SxE methods by CALUX assay and HRGC/MSSample No.
Ultrasonic Extraction (UsE)
Soxhlet Extraction (SxE)
Ratio
HRGC/MS CALUX- CALUX-SxE/CALUX CALUX Ave. S.D. C.V. CALUX
2ndSxEUsE /SxE
HRGC/MS
1st10.55
1.5
1.0 0.70
67
1.5
0.7
20.27
3.4
1.8
2.2
120
3.0
0.6
3
1.5
0.31
0.93 0.87
94
1.3
0.7
40.08
1.4
0.75 0.94 126
1.10.75
1.0
1.6
1.3 0.36
28
1.2
1.162621243.3
1420
111.2
1.876.6
9.7
8.2
2.2
27
12
170.7
0.78202221
1.6
7.4
2116
1.0
1.3
928
3129
2.4
8.1
24
19
1.2
1.3
Comparison of CALUX and HRGC/MS

HRGC/MS (pg-TEQ/g)

1200
1000

y = 0.5388x
R² = 0.9966

800
600

y = 0.3068x
R² = 0.9998soilfly ash

400
2000010002000
3000
CALUX (pg-TEQ/g)

4000

Fig. 1 Comparison of CALUX-TEQ and WHO-TEQ for four soil samples andtwo fly ash samples obtained from Hiyoshi Corporation in Japan.

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Soxhlet Extraction (pg-TEQ/g)

CALUX-TEQ
30.0
y = 0.8879x
R² = 0.956

25.0
20.0
15.0
10.0
5.0
0.0

0.0

5.0

10.0
15.0
20.0
25.0
30.0
Ultrasonic Extraction (pg-TEQ/g)

35.0

Fig. 2 Comparison of ultrasonic and soxhlet extraction by CALUX assay for the nine samples
collected from a municipal waste landfill in China

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