IMPLEMENTATION OF THE CALUX BIOASSAY FOR THE ANALYSIS OF PCDD/F’SAND DIOXIN-LIKE PCB’S IN ATMOSPHERIC DEPOSITION SAMPLES FROM THEFLEMISH MONITORING NETWORKVandermarken T1, Van Langenhove K1, Elskens M1, Desmedt M2, Roekens E2, Denison MS3, Baeyens W1,Croes K1,41

Vrije Universiteit Brussel, Department of Analytical and Environmental Chemistry (ANCH), Pleinlaan 2, 1050
Brussels, Belgium, 2 VMM, Flemish Environment Agency, Kronenburgstraat 45/3, 2000 Antwerp, Belgium, 3
Department of Environmental Toxicology, University of California, Davis, CA 95616, USA, 4Ghent University
Hospital, Study Centre for Carcinogenesis and Primary Prevention of Cancer, Ghent, Belgium
Introduction
Although the risks of PCDD/Fs and PCBs are well known and a lot of research has been done during the recentyears, these compounds are still environmental pollutants of major concern. In Flanders, the Flemish
Environment Agency (VMM) has a monthly measurement program for the monitoring of atmosphericdepositions of PCDD/Fs and PCB 126 in different locations in Flanders. Since the beginning of the measurement
campaign in Flanders in 1995, concentrations have decreased drastically. However, in certain locations thewarning limit for agricultural and residential areas, set by the VMM based on tolerable intake values, VDI
guideline 2090/1 and a model study by VITO, of 21 pg TEQ m-² day-1 for mean monthly values1 (sum ofPCDD/Fs and dioxin-like PCBs) is regularly exceeded. Since PCDD/Fs and dioxin-like PCBs (dl-PCBs) arepersistent in the environment, can accumulate in the fat tissue of animals and humans and have hormone
disrupting properties, it is important to have a reliable, fast and inexpensive method to monitor, on a regularbasis, the concentration of these pollutants. Until now, only GC-HRMS methods are available for thequantification of PCDD/Fs and dl-PCBs in atmospheric deposition samples2, 3. In this study, a new method forthe analysis of PCDD/Fs and dl-PCBs in atmospheric deposition samples with the CALUX bioassay wasdeveloped, optimized and validated. More than 100 samples from the Flemish monitoring network wereanalyzed with both CALUX and GC-HRMS to define the correlation between both techniques and to investigate
whether the CALUX bioassay can be used instead of or in addition to GC-HRMS for the analysis of PCDD/Fsand dl-PCBs in atmospheric deposition samples in Flanders.
Materials and methods
The sample preparation and bioassay measurement protocols were described previously by Croes et al., 20114
Briefly, the method consists of 4 steps: filtration, extraction, clean up and CALUX bioassay analysis. To avoid
the use of large amounts of toxic solvents, new techniques were used for filtration and extraction: a C18 filter
was used instead of a liquid/liquid extraction and an Accelerated Solvent Extractor (ASE) was used instead of
the traditional soxhlet extraction. After pre-oxidation of the sample extract with concentrated sulphuric acid,clean up was done using a multi-layer silica gel column coupled to a carbon column. The PCDD/F and PCB
fractions were finally analyzed with the H1L7.5c1 and/or the H1L6.1c3 mouse hepatoma cell lines5,6.
Results and discussion
In this study, a new CALUX method was developed and validated. The limit of quantification was 1.4 pg
CALUX-BEQ m-² day-1 for the PCBs and 5.6 pg CALUX-BEQ m-² day-1 for the PCDD/Fs, when using the newsensitive H1L7.5c1 cell line. The GC-HRMS recovery of spiked atmospheric deposition samples was for all 13C
PCDD/F congeners between 55 and 112%, with a mean recovery of 90%. CALUX recoveries of spiked
procedural blanks were between the accepted ranges of 80-120%. Repeatability and reproducibility weresatisfactory and no interferences from metals were detected.
For more than 100 atmospheric deposition samples from industrial, agricultural, residential and wildlife areas,collected between September 2009 and May 2010, PCDD/Fs and dl-PCBs were analyzed by the CALUX
bioassay. BEQ determination was done in the optimum dilution range, around the EC50 value of the sample
dose-response curve. In contrast to biological samples (i.e. blood7 and milk8), for environmental samples it is not

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possible to establish a full dose dilution curve from a pooled sample extract that can be used as a benchmark forthis type of sample matrix for the determination of an optimum dilution factor. Therefore, it was decided that forevery atmospheric deposition sample a (full dose) dilution curve would be generated for both the dioxin-like
PCB and PCDD/F fractions and these would be used to calculate the CALUX-BEQ concentration.
In Table 1, GC-HRMS and CALUX results are given for subset of 19 samples, for both the PCDD/Fs and the dlPCBs, from 5 different agricultural or residential areas in Flanders. The samples were taken during 4 sampling
campaigns. To compare the GC-HRMS and CALUX results and to evaluate the number of samples that wereabove the GC-HRMS warning limit, the sum of PCDD/Fs and dl-PCBs for GC-HRMS measurements wascalculated according to the following formula (equation 1), where it was assumed that PCB 126 accounts for 70% of the total dl-PCB toxic equivalent9.
Σ PCDD/Fs and dl-PCBs GC-HRMS = [WHO-TEQ PCDD/Fs]GC-HRMS + ([WHO-TEQ PCB126]GC-HRMS / 0.7)
Equation 1: calculation of the TEQ for the sum of PCDD/Fs and dl-PCBs with GC-HRMS
From Table 1, it is clear that dl-PCB values were generally lower with the CALUX bioassay than with GCHRMS, except for very low PCB concentrations (close to the limit of quantification; i.e. Beerse 1 fromcampaign 3). For the PCDD/F fraction, CALUX values were higher compared to the GC-HRMS WHO-TEQvalues. CALUX/GC-HRMS ratios ranged from 1.2 to 5.5, with a median ratio of 2.0. For some sampling
locations, the ratios were relatively constant (i.e. Roeselare 6), while for other locations (i.e. Beerse 1) morevariation was found during different sampling campaigns. This can be due to other interfering compounds thatare present in the atmospheric deposition samples and can vary in concentration level over time (seasonaleffects). For the sum of PCDD/Fs and dl-PCBs, ratios between 0.7 and 5.1 were found, with a median value of
1.5.
Overall, for most samples a satisfactory correlation was found between the CALUX and GC-HRMS results, forboth the PCDD/Fs and dl-PCBs. There was also a close agreement between the CALUX-BEQ values fromatmospheric deposition samples taken in duplicate from the same sampling location. This shows that theCALUX bioassay can be implemented in the Flemish GC-HRMS measurement network. Especially as ascreening technique, for identifying possible contaminated new measurement locations and for determining themost suitable geographical location for the quantification of PCDD/Fs and dl-PCBs in atmospheric deposition
samples (taking into account distance and wind direction from a point source), CALUX can be a valuable tool.
Acknowledgements
This work was financially supported by the Flemish Environment Agency. The authors are grateful to M.
Denison and the other members of the University of California-Davis (USA) for generously providing ourlaboratory with the H1L6.1c3 and the H1L7.5c1 cell lines which were developed with funding from a Superfund
Research Program grant (ES04699) from the National Institute of Environmental Health Sciences. The authors
also acknowledge A. Kotz and R. Malisch (European Union Reference Laboratory for Dioxins and PCBs in
Food and Feed, Germany) and the company SGS (Melsele, Belgium) for the GC-HRMS analysis.
References:
1. Desmedt M, Roekens E, De Fre R, Cornelis C, Van Holderbeke M. (2008); Organohaogen. Compounds 70:
1232-12352. Man R, Pingan P, Sukun Z, Liping Y, Bixian M, Guoying S, Jiamo F. (2006); Chin J Anal Chem 34(1), 16−20
3. Moon HB, Lee S-J, Choi H-G, Ok G. (2005); Chemosphere. 58: 1525-1534
4. Croes K, Van Langenhove K, Elskens M, Desmedt M, Roekens E., Kotz A, Denison MS, Baeyens W. (2011);
Chemosphere 82 (5): 718-724
5. Denison MS, He G, Baston DS, Tsutsumi T. (2008); Organohalogen Compounds 70: 772-775
6. He G, Tsutsumi T, Zhao B, Baston DS, Zhao J, Heath-Pagliuso S, Denison MS. (2011); Toxicological
Sciences (submitted)

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7. Croes K, Van Langenhove K, Den Hond E, Bruckers L, Colles A, Koppen G, Loots I, Nelen V, Schoeters G,
Nawrot T, Van Larebeke N, Denison MS, Vandermarken T, Elskens M, Baeyens W. (2011); Organohalogen
Compounds 73 (this symposium).
8. Croes K, Vandermarken T, Colles A, Bruckers L, Koppen G, Govaerts E, Nelen V, Van de Mieroop E,Schoeters G, Van Larebeke N, Denison MS, Kotz A, Van Langenhove K, Elskens M, Baeyens W. (2011);
Organohalogen Compounds 73 (this symposium).
9. Desmedt M, Roekens E. (2011); Organohalogen Compounds 73 (this symposium).

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Location

Campaign

GC-HRMSCALUX

sum PCDD/Fs
sum PCDD/Fs
PCDD/Fs dl-PCBs& dl-PCBs& dl-PCBs
Merksem
2
4,7
2,1
6,7
1,5
8,3
1,4
0,7
1,1
Merksem
53,9
3,1
6,8
1,5
8,3
1,70,51,0
Beerse 11142,178
2,880
5,5
1,3
4,8Beerse 1
2
139,6
15
3,4
19
0,30,71,2
Beerse 1
38,70,8
47
3,2
51
5,5
3,9
5,1
Menen 10
14,8291716
33
3,50,60,7
Menen 10
2
3,3
1,8
7,6
2,1
9,8
2,3
1,2
1,7
Menen 103272,4
29
3,7
1,3
3,0
7,2
1,8
9,8
Menen 10
323
1,525
3,2
0,82,5
Roeselare 6
2
15
6,7
22
1,62,3
1,6
9,32,913
Roeselare 6
2
12
5,9
17
1,22,01,3
Roeselare 6
59,93,1
13
1,4
1,2
1,2
6,92,611Roeselare 65103,4
14
1,51,31,3
Stabroek
1131,815
2,00,8
1,5
6,72,3
10
Stabroek
1132,015
2,00,91,5
Stabroek
2
4,20,9
5,5
15
1,9
17
3,52,13,0Stabroek
3392,541
2,6
1,52,415
1,717
Stabroek
3222,524
1,5
1,51,4
Stabroek
5
3,6
1,0
5,0
9,2
1,7
11
2,6
1,72,2
Table 1: GC-HRMS and CALUX results for the PCDD/Fs and dl-PCBs from a subset of 19 samples, collected during 4 different samplingcampaigns on 5 different locations in Flanders. The numbers in red were higher than the GC-HRMS warning limit.
PCDD/Fs

Organohalogen Compounds

PCB126

sum PCDD/Fs &dl-PCBs
7,78,3
17
27
9,8
46
5,9

CALUX/ GC-HRMS ratio

PCDD/Fs

dl-PCBs

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