ANALYSIS ON PCDD/Fs IN SOIL AROUND WASTE INCINERATIONPLANTS IN CHINA USING CALUX ASSAY WITH HRGC/HRMS
Du YZ1, Yan JH1, Chen T1, Lu SY1, Li XD1, Cen KF1, Nakamura M2, Huang JQ21

State Key Laboratory of Clean Energy Utilization, Zhejiang University, Hangzhou, PR China
2
Hiyoshi Ecological Services Co., Kitanoshyo 908, Omihachiman, Shiga 523-0806, Japan

Introduction
Polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and dioxin-like
polychlorinated biphenyls (DL-PCBs) are a group of toxic related compounds, known as dioxins1.
Dioxins are not produced intentionally, but they are by-products generated from processes when heat isapplied to substances containing carbon, oxygen, hydrogen and chlorine2. The major and importantsource of these compounds at present is waste incineration, with most being formed in combustion
processes or cooling stage of flue gas1-4. Dioxins is then emitted to the ambient air without being fully
captured by flue gas purification equipment, and may accumulate in the soil in surrounding
environment. Recently, a great number of waste incineration plants have been constructed in China,including municipal solid waste and hazardous waste incinerators. Moreover, the toxicity and pollution
condition of dioxins is arousing more and more attention from the common people. The monitoring of
dioxins in soil around waste incinerators must to be conducted at present. In order to understand thedioxins levels in the surrounding soil of some typical waste incineration plants, one of analysis
methods, that is CALUX assay, was carried out to analyze the concentrations of PCDD/Fs in this study.
Additionally, TEQs of PCDD/Fs obtained by the CALUX assay were compared with WHO-TEQ
obtained by traditional chemical analysis--HRGC/HRMS.
Materials and methods
The soil samples in this work were divided into two part. The first five soil samples, which are of highconcentration, were obtained from Hiyoshi Corporation in Japan. The left twenty-four sample, which
are of respectively low concentration, were collected from three different kinds of municipal solid or
hazardous waste incineration plants in Zhejiang Province, China. The second part of soil samples werecollected by inserting a cylindrical steel corer down to a 10cm depth. Approximately 1.5 kg of soil was
taken at each site. The soils were subsequently dried in a ventilated room. Then they were ground andpassed through a 2-mm sieve. About 500 g soil of each sample was homogenized through a 60-meshsieve, and was refrigerated until analysis.
For HRGC/HRMS analysis, about 10g (dry matter) of soil samples were taken out for extraction,clean-up procedure and analytical determination, described as the previous research5. A fully automatedASE 300 system (Dionex, Sunnyvale, CA, USA) was used for sample extraction. Each sample wasspiked with a mixture of 13C-labelled PCDD/Fs compound stock solution (5μl) and clean-up standard(5μl) before extraction. The extracts from ASE were subsequently passed through multilayer silica gelcolumn following USEPA Method 1613. The extracts were blow down to 20μl by nitrogen (N2), and5μl internal standard solution were added before sample were subjected to PCDD/Fs analysis by using
HRGC/HRMS (JEOL JMS-800D) with a DB-5MS column (60m×0.25mm×0.25µm).
Soil samples (3.50-3.54g) dissolved in 10ml 20%methanol/toluene, and then the solution was shaken

Organohalogen Compounds

Vol. 72, 88-91 (2010)88and ultrasonic extracted for 5min, which were prepared for CALUX assay. The extraction waspassed through a celite column, and then the above operations were conducted two times. The
mixed solution was further loaded onto an acid-silica gel and XCARB (XDS Inc., USA) bigeminal
columns. The DL-PCBs and PCDD/Fs fractions were then eluted with different solution only onto
XCARB column respectively, and the PCDD/Fs fraction was selected for CALUX assay in thisstudy. The CALUX assay was carried out using a recombinant H1L6.1 cell line. The cells wereseeded on 96-well microplates, and after 24h of incubation, cells were exposed in duplicate to thepurified sample extracts and 2,3,7,8-TCDD standard solution for 20-24h of incubation. The
luciferase activity was measured using a Lucy I luminometer (Antos, Salzburg, Austria).
Results and discussion
The TEQ of PCDD/Fs determined by CALUX assay and HRGC/HRMS with WHO-TEF(2005)
are written as “CALUX-TEQ” and “WHO-TEQ”, respectively. CALUX-TEQ and WHO-TEQ offive soil samples from Hiyoshi Corporation were 20.8～7795.2 pg-TEQ/g and 9.7～6760.0
pg-TEQ/g respectively (not shown in this paper), as shown in Fig.1. These results show thatCALUX-TEQ and WHO-TEQ are highly correlated in soil samples. The ratio between
CALUX-TEQ and WHO-TEQ were 1.4±0.7, so generally, CALUX-TEQ showed higher values
than WHO-TEQ6.
WHO-TEQ of the twenty-four soil samples collected from three waste incineration plants in China
were in the range of 1.72 to 31.44 pg-TEQ/g (not shown in this paper), with a mean value of 6.09pg-TEQ/g. Schuhmacher et al.7 investigated the baseline level of PCDD/Fs in soil in Catalonia(Spain), and PCDD/Fs levels ranged from 0.13 to 24.2 pg I-TEQ/g, with a mean value of 1.68 pg
I-TEQ/g. In addition, the WHO-TEQ values observed in the present study are basically consistent
with those found in Barcelona (Spain; 6.78～16.62 pg I-TEQ/g)8 and Buchon (Korea; 1.25～74.98 pg I-TEQ/g)9. Furthermore, CALUX-TEQ of the twenty-four soil samples were in the rangeof 1.71 to 44.23 pg-TEQ/g, and CALUX-TEQ and WHO-TEQ values are compared graphically inFig.2. It is obvious that CALUX-TEQ is significantly correlated with WHO-TEQ of thetwenty-four samples from China, and the correlation coefficient is 0.7639, which is lower thanthat of the five samples from Hiyoshi Company. Notably, maybe PCDD/Fs concentration in thesamples from Hiyoshi Company is extremely higher than that from China, and the concentration
distributes between 9.7 to 6760.0 pg-TEQ/g, which is more dispersive, while the distribution from
China is centralized in the range of 1.72～31.44 pg-TEQ/g. Therefore, the correlation of thesecond part samples (China) is not very satisfactory, but the CALUX assay for PCDD/Fs
determination has been increasingly recognized as a useful tool. Moreover, the development of acorrelation database between WHO-TEQ and CALUX-TEQ for soil samples is required in thefuture.
The CALUX assay is conducted by the reaction of specific cell line with PCDD/Fs congeners, soCALUX-TEQ values depend on the biological responsiveness to specific PCDD/Fs isomers. To
examine the factors affecting the discrepancy found by TEQ determined by bioassay and chemical
analysis, relative potency (REP) values for individual PCDD/Fs isomers were applied (the data
were shown in another paper)10. REP-TEQ was calculated by multiplying the concentration ofeach isomer measured by HRGC/HRMS with its CALUX-REP. Interestingly, CALUX-REP was

Organohalogen Compounds

Vol. 72, 88-91 (2010)89found to be close to WHO-TEQ, except that the REP values of 1,2,3,7,8-PeCDF,
2,3,4,6,7,8-HxCDF, 1,2,3,4,7,8,9-HpCDF and OCDF were much higher than WHO-TEF values. Itis indicated that biological responsiveness of CALUX assay is much more active to PCDFs, which
is a possible factor of differences between CALUX-TEQ and WHO-TEQ. Furthermore, it isspeculated that the presence of other AhR agonisits is another possible factor of discrepancy.
Polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/Fs) and a few polychlorinatednaphthalene (PCNs) congeners also have dioxin-like activities11.
PCDD/Fs homologues were shown in Table 1. OCDD was the predominant homologue, and the othermajor homologues are HpCDD, HpCDF and OCDF. Due to the soil samples from different kinds ofwaste incineration plants, the ratios of PCDFs to PCDDs in soil samples vary greatly, which are theimportant indicator to ascertain the possible reaction routes for PCDD/Fs formation. For example,[PCDF]/[PCDD] ratios of S20～S24 from the same plant are above one, which indicates that the mainroute for PCDD/Fs formation is de novo synthesis, and when [PCDF]/[PCDD] ratios of S11～S19
from the same plant are below one, the main route is precursor condensation.
Acknowledge
This research work was financially supported by Hiyoshi Corporation, Japan.
Reference
1. B.R. Stanmore, (2004), Combust. Flame, 136, 398–427.
2. Government of Japan, Dioxins, 2009.
3. H. Fiedler, (2007), Chemosphere, 67, S96.
4. E. Finocchio, G. Busca, M. Notaro, (2006), Appl. Catal., B62, 12–20.
5. Li XD, Yan M, Chen T, Lu SY, Yan JH, Cen KF, (2010), Journal of Hazardous Materials, 179, 783-789.
6. US Environmental Protection Agency, Test methods for evaluating solid waste, physical/chemical methods. USEPA, 2007.
7. M. Schuhmacher, M.C. Rodriguez-Larena, M.C. Agramunt, J. Diaz-Ferrero, J.L. Domigo, (2002), Chemosphere, 48, 187-193.
8. M. Schuhmacher, S. Granero, J. Rivera, L. Muller, J.M. Llobet, J.L. Domigo, (2000), Chemosphere, 40, 593-600.
9. J.E. Oh, S.D. Choi, S.J. Lee, Y.S. Chang, (2006), Chemosphere, 64, 579-587.
10. Brown D.J., Chu M. van, Overmerir L., Chu A., Clark G.C., (2001), Organohalogen Compd., 53, 211-214.
11. Jun Hasegawa, Keerthi S. Guruge, Nobuyasu Seike, Yuji Shirai, Toshiaki Yamata, Masafumi Nakamura, Hiroshi Handa,
Noriko Yamanaka, Shigeru Miyazaki, (2007), Chemosphere, 69, 1188-1194.

PCDD/Fs
8000
HR GC/MS(pg-TEQ/g)

7000
6000
5000
y = 1.7934x
4000

2

R = 0.9956

3000
2000
1000
00500

1000

1500

2000

2500

3000

3500

4000

CALUX (pg-TEQ/g)

Organohalogen CompoundsVol. 72, 88-91 (2010)

90Fig. 1 Comparison of CALUX-TEQ and WHO-TEQ for five soil samples
from Hiyoshi Corporation in Japan.

PCDDs /PCDFs

HRGC/MS(pg-TEQ/g)

35y = 0.5713x

30

2R = 0.7639

252015105001020305040

ZJU CALUX (pg-TEQ/g)

Fig. 2 Comparison of CALUX-TEQ and WHO-TEQ for twenty-four soil samples
collected from three waste incineration plants in China.
Table 1 PCDD/Fs homologues in soil samples colleted from China
S1S2S3S4S5S6S7S8S9S10S11S12TeCDDPeCDD0.15
0.77

0.25
0.95

0.13
0.51

0.25
0.69

0.00
0.00

0.17
1.11

0.15
1.01

0.15
1.18

0.09
1.33

0.06
0.97

1.76
9.15

0.34
2.76

HxCDD

2.58

2.23

4.32

2.99

3.24

5.77

5.28

6.99

8.13

3.16

44.41

20.15

HpCDD

4.10

5.03

37.62

18.82

26.90

19.40

14.20

24.60

35.60

8.54

684.54355.25

OCDD

61.68

78.11

1392.68

776.79816.79

61.20

65.80

62.20

71.10

39.50

27182.50

9840.81

TeCDF

0.97

1.46

1.15

1.56

1.23

1.09

1.09

0.88

1.07

0.78

1.17

0.15

PeCDF2.43

2.49

1.73

3.00

1.62

3.72

3.98

3.43

5.06

1.87

2.83

0.42

HxCDF

4.35

4.52

4.16

5.28

4.07

12.41

11.74

15.21

21.21

5.37

4.19

1.13

HpCDF

4.23

4.35

4.41

5.81

4.29

21.75

20.12

27.10

32.54

9.57

7.95

1.21

OCDF

5.06

8.23

7.50

3.67

0.00

10.70

11.20

14.50

22.30

6.99

16.96

6.45

S13

S14

S15

S16

S17

S18

S19

S20

S21

S22

S23

S24

TeCDD
PeCDD

0.31
0.49

0.65
3.84

0.11
0.45

0.41
2.45

0.39
1.83

0.22
1.43

0.69
3.71

0.09
0.00

0.271.82

0.00
0.53

0.21
0.18

0.03
0.50

HxCDD

3.99

15.61

2.89

9.60

9.49

8.53

16.74

0.81

1.02

0.96

1.86

1.29

HpCDD

108.06130.08

73.75

99.46

127.45168.90104.20

6.95

4.26

10.68

4.22

9.00

OCDD

11317.50

4956.198391.784868.265424.015965.92

1066.52771.71226.051463.2744.04

1019.55

TeCDF

2.01

2.37

1.41

2.04

0.70

0.18

1.65

4.51

1.01

5.95

1.10

3.59

PeCDF

3.55

6.63

3.03

4.48

1.59

0.57

5.31

2.38

6.54

6.40

3.46

3.53

HxCDF

8.96

11.85

7.97

8.45

2.63

1.22

8.85

5.97

7.89

13.91

6.97

7.58

HpCDF

15.11

14.81

14.21

14.07

5.23

1.66

12.81

7.28

25.29

19.09

10.96

10.64

OCDF

8.10

2.53

4.43

3.13

3.80

3.56

0.99

9.55

221.54350.67

7.71

14.19

Organohalogen Compounds

Vol. 72, 88-91 (2010)91