Comparison of XDS-CALUX® to HR GC/MS
Full method at
4435 - 1 Revision 0
September 2007
METHOD 4435
METHOD FOR TOXIC EQUIVALENTS (TEQS) DETERMINATIONS FOR DIOXIN-LIKE
CHEMICAL ACTIVITY WITH THE CALUX® BY XDS BIOASSAY
SW-846 is not intended to be an analytical training manual.
Therefore, method procedures are written based on the assumption that they will
be performed by analysts who are formally trained in at least the basic
principles of chemical analysis and in the use of the subject technology. In
addition, SW-846 methods, with the exception of required method use for the
analysis of method-defined parameters, are intended to be guidance methods
which contain general information on how to perform an analytical procedure or
technique which a laboratory can use as a basic starting point for generating
its own detailed Standard Operating Procedure (SOP), either for its own general
use or for a specific project application. The performance data included in
this method are for guidance purposes only, and are not intended to be and must
not be used as absolute QC acceptance criteria for purposes of laboratory
accreditation.
1.0 SCOPE AND APPLICATION
1.1 Method 4435 is a bio-analytical procedure that is based on the
mechanism of action of dioxin-like chemicals which allows for the determination
of the relative toxic potential of sample extracts containing these chemicals
and the resulting potency values are expressed as Toxic Equivalents (TEQs).
Method 4435 is a bio-analytical method that is based on the ability of dioxin
and related chemicals to activate the Ah receptor (AhR), a chemical-responsive
DNA binding protein that is responsible for producing the toxic and biological
effects of these chemicals. Measurement of the level of activation of
AhR-dependent gene expression by a chemical or chemical extract provides a
measure by which to estimate the relative potency and toxic potential of these
chemicals and/or extracts with resulting values expressed as Toxic Equivalents
(TEQs).
Xenobiotic Detection Systems (XDS web site: www.dioxins.com) has a commercially
available genetically engineered cell line that contains the firefly luciferase
gene under trans-activational control of the AhR (U.S. patent # 5,854,010).
This cell line can be used for the sensitive detection and relative
quantification of AhR agonists and agonist activity of complex mixtures. Our
term for the in vitro assay is the XDS Chemical-Activated Luciferase Expression
or CALUX®
by XDS assay. The most widely studied class of compounds that activate this
system is the polychlorinated diaromatic hydrocarbons (PCDH), such as
2,3,7,8-tetrachlorodibenzo-p-dioxin
(2,3,7,8-TCDD, dioxin). The relative toxic and biological potency
of many PCDH compounds are quantified and expressed relative to that of
2,3,7,8-TCDD, since this is one of the most potent activators of AhR-mediated
effects, including gene transcription. This relative quantification approach
generates overall potency values known as Toxic Equivalents (TEQs) and the
results obtained from the CALUX® by XDS assay provide a measure of TEQs in a sample. By using XDS's
sample processing procedures and an affinity column (U.S. Patent # 6,720,431) polychlorinated
biphenyls (PCBs) can be separated from chlorinated dioxins/dibenzofurans (PCDDs/PCDFs)
making it possible to determine what portion of the total TEQs of a sample is due
to each of these classes of compounds. XDS has termed this the Dioxin/Furan-
and PCBspecific(DIPS) analysis or the DIPS-CALUX bioassay for dioxin-like
chemicals.
The AhR-dependent mechanism
of the toxic and biological effects of dioxin-like chemicals and the basis of
the CALUX® by XDS bioassay measurement and estimate of TEQ is [shown
above] (Denison et al., 2004). The AhR receptor complex is capable of binding dioxins, furans,
PCBs and other dioxin-like compounds. Once these chemicals bind to the AhR, the
complex migrates into the nucleus where it specifically binds to the ARNT protein.
The resulting chemical: AhR:ARNT complex then binds to a specific DNA sequence,
the Dioxin Responsive Element (DRE), which is present upstream from many genes
including that of CYP1A1, and this binding stimulates expression of the
adjacent gene. In the case of the CALUX® by XDS assay, a plasmid containing four DREs
immediately upstream of the firefly luciferase reporter gene was stably transfected into the mouse
Hepa1c1c7 cell line to produce the recombinant cell line H1L6.1c3 (6.1 cells).
This transformed cell line responds to toxic PCDDs, PCDFs and PCBs, and high
molecular weight polynuclear aromatic hydrocarbons (PAHs) with the
dose-dependent induction of firefly luciferase (Garrison et al., 1996; Denison et al., 2002, 2004;
Ziccardi et al., 2002; Han et al., 2004). Comparison of these results to
a 2,3,7,8-TCDD standard curve for induction allows for determination of the
TEQs in a given sample.
By using XDS sample processing methods (U.S. patent # 6,720,431)
it is possible to separate polyhalogenated biphenyls from polyhalogenated
dioxins/dibenzofurans present in the same sample. Using this DIPS-CALUX® bioassay it is possible to
determine the portion of the total TEQ activity in a given sample that is due
to each of these classes of compounds (Brown et.,al 2002).
NOTE: The bioassay testing product listed in this method has been
submitted to EPA, evaluated by the Agency, and found to meet the performance specifications
necessary for inclusion in SW-846. As additional testing products are evaluated
by EPA and found to provide equivalent performance, information will be made
available by the Office of Solid Waste regarding those testing products that
are capable of meeting the performance specifications in this method
(See http://www.epa.gov/epaoswer/hazwaste/test/pdfs/kits.pdf). However, this procedure will not be revised
solely to include information on additional testing products. Descriptions and
materials lists for products relevant to this method are provided in Table 3
and are given in the manufacturer’s literature.
1.2 The CALUX® by
XDS method for TEQ estimation of dioxin-like chemicals. The CALUX® by XDS method is a
relatively rapid screening method capable of estimating the Toxic Equivalents
(TEQs) concentration for dioxin-like chemicals in a sample. The sample is extracted in an organic solvent
and fractionated through the sample processing procedure. An extract that
contains the halogenated dioxins/furans is separated from an extract containing
the halogenated biphenyls. These extracts are applied to monolayers of our
H1L6.1c3 cells and the amount of luciferase induction is measured after 20 to 24
hours. A standard dilution series of 2,3,7,8-TCDD is included on each plate of
cells.
Estimation of dioxin/2,3,7,8-TCDD-like TEQ activity present in the
sample extract is performed by extrapolation to the 2,3,7,8-TCDD standard curve
by least squares estimates with the 4 parameter Hill Equation.
There are three modes by which the DIPS-CALUX bioassay is
performed. These are the screening mode with historical recovery, screening
mode surrogate recovery, and the semiquantitative mode. The screening mode
involves the analysis of a single aliquot of the sample and recovery is estimated
from the mean of historical recoveries that have been obtained for soils/sediment
samples. This is considered to be acceptable as the variability of recoveries
for soils/sediment samples has been relatively small (76.2 +/- 8.5%). Using
this mode would indicate whether a sample needed to be further analyzed by
either the semi-quantitative mode or by chemical analysis. The screening mode
surrogate recovery, involves processing two aliquots of the sample, the first for analysis in the DIPS-CALUX
bioassay and the second used for the surrogate spike with radiolabeled 2,3,7,8-
TCDD to estimate recovery. The semiquantitative mode involves analyzing three
aliquots of the sample in the DIPS-CALUX bioassay and a fourth aliquot of the
sample used for determination of recovery with radiolabeled 2,3,7,8- TCDD. The
cost of sample analysis is dependent upon which mode of the DIPS-CALUX bioassay
is used for estimation of the levels of sample contamination.
1.3 Toxic Equivalents (TEQs):
The concept of Toxic Equivalents (TEQs) has been promulgated by
the World Health
Organization to provide a means of quantifying for risk assessment
purposes the toxicity of a family of chemicals with a similar overall mechanism
of toxicity (Van den Berg,
1998). The family of dioxin-like chemicals (PCDHs) within this group
includes 7 chlorinated dibenzo-p-dioxin congeners with 4 to 8 chorines on the
molecule, 10 chlorinated dibenzofuran congeners with 4 to 8 chlorines on the
molecule, and 12 chlorinated biphenyls with 4 to 10 chlorines on the molecule.
16.0 REFERENCES
Brown, D. J., Nakamura, M., Chu, M.D., Denison, M.S., Murata, H.,
and Clark, G.C.
(2002). "Recovery determinations for bioassay analysis:
Condierations and results."
Organohalogen Compounds 58: 357-360.
Brown, D. J., Van Overmeire, I., Goeyens, L., Chu, M.D., Denison,
M.S., and Clark, G.C.(2002). "Elimination of interfering compounds in
preparation for analysis by an Ah receptor based bioassay." Organohalogen
Compounds 58: 401-404.
Clark, G., V. Garry, et al. (2002). "Relationships between
exposure to dioxin-like
chemicals, testosterone levels, and sex of the children of
pesticide applicators."
Organohalogen Compounds 56: 73-76.
Clark, G. C., Brown, D.J., Seidel, S.D., Phelan, D., Denison, M.S.
(1999).
"Characterization of the CALUX and GRAB bioassays for
sensitivity and specificity in
detection of phamacological agents that activate the Ah Receptor
signaling system."
Organohalogen Compounds 42: 309-312.
Clark, G. C., Chu, M.,
Touati, D., Rayfield, B., Stone, J., Cooke, M. (1999). "A Novel
Low-Cost Air Sampling
Device (AmbStack Sampler) and Detection System (CALUX
Bioassay) for Measuring Air
Emissions of Dioxin, Furan, and PCB on a TEQ Basis
Tested With a Model
Industrial Boiler." Organohalogen Compounds 42: 309-312.
Denison, M. S., Nagy, S.R.,
Clark, G.C., Chu, M., Brown, D.J., Murata, H., Shan, G.,
Sanborn, J.R., and Hammock,
B.D. (2001). "Bioanalytical approaches for the Detection
of Dioxin and Related
Halogenated Aromatic Hydrocarbons." Organohalogen
Compounds 45.
Denison, M. S., Seidel,
S.D., Ziccardi, M., Rogers, W.J., Brown, D.J., and Clark, G.C.
(1999). "Ah
receptor-based bioassays for dioxins and related chemicals: Applications
and limitations."
Organohalogen Compounds 40: 27-30.
Denison, M.S., Zhao, B.,
Baston, D.S., Clark, G.C., Murata, H. and Han, D.-H. (2004)
Recombinant Cell Bioassay
Systems for the Detection and Relative Quantitation of
Halogenated Dioxins and
Related Chemicals, Talanta 63: 1123-1133.
Denison, M.S. Nagy, S.R.,
Ziccardi, M., Clark, G.C., Chu, M., Brown, D.J., Shan, G.,
Sugawara, Y., Shirley J.
Gee, S.J., James Sanborn, J. and Hammock, B.D. (2002)
Bioanalytical approaches
for the detection of dioxin and related halogenated aromatic
hydrocarbons, in:
Technology-Driven Biomarkers Development and Application in
Environmentally-Associated
Diseases, Wilson, D. and W. Suk, W., eds., pp. 483-494,
Lewis Press, Boca Raton,
FL.
Garrison, P.M., Tullis,
K., Aarts, J.M.M.J.G., Brouwer, A. and Giesy, J.P. and Denison,M.S. (1996)
Species-specific recombinant cell lines as bioassay systems for the detection
of 2,3,7,8-tetrachlorodibenzo-p-dioxin-like chemicals, Fund. Appl. Toxicol.
30,194-203.
Han, D.-H., Nagy, S.R.
and Denison, M.S. (2004) Comparison of recombinant cell Bioassays for the
detection of Ah receptor agonists, Biofactors 20, 11-22.
Han, D., Nagy, S.R.,
and Denison, M.S. (2002). "Recombinant cell lines for the detection of
dioxins and Ah Receptor ligands- Not all assays are created equal."
Organohalogen Compounds 58: 421-424.
Ziccardi, M.H.,
Gardner, I.A. and Denison, M.S. (2002) Application of the luciferase
recombinant cell culture
bioassay system for the analysis of polycyclic aromatic
hydrocarbons, Environ.
Toxicol. & Chem. 21, 2027-2033.
Windal, I., Dennison,
M. S, Birnbaum L. S., Van Wouwe, N., Baeyens, W. Goeyens L.
(2005). “Chemically
Activated Luciferase Gene Expression (CALUX) Cell Bioassay
Analysis for the Estimation
of Doxin-Like Activity: Critical Parameters of the CALUX
Procedure that Impact Assay
Results, Environ.” Sci. Technol., 3, 7357-7364.
Van den Berg et al
(2006). “The 2005 World Health Organization Reevaluation of
Human and Mammalian Toxic
Equivalency Factors for Dioxins and Dioxin-Like
Compounds.” Toxicological
Sciences 93(2):223-241.
www.dioxins.com/pdf/environment/environment09.pdf
How to measure dioxins in a smokestack using CALUX
Analysis P006
ORGANOHALOGEN
COMPOUNDS
Vol.40 (1999)
pp. 79-82
A Novel
Low-Cost Air Sampling Device (AmbStack Sampler) and Detection System (CALUX
Bioassay) for Measuring Air Emissions of Dioxin, Furan, and PCB on a TEQ Basis
Tested With a Model Industrial Boiler
George C. Clark
1, Michael Chu 1, Dahman Touati 2, Barry Rayfield 3, Jon Stone 4, and Marcus
Cooke 5
Affilitations: 1
Xenobiotic Detection Systems, Durham, NC, 2 Arcadis, Durham, NC, 3 Kilkelly Associates,
Raleigh, NC, 4 URG, Chapel Hill, NC, 5 Cooke Companies International, Chapel
Hill, NC
Introduction
The analysis of
polychlorinated dibenzo-p-dioxin (PCDD) and polychlorinted dibenzofuran (PCDF) in
gaseous samples is very labor intensive, and expensive. Regulatory reporting
usually requires a sampling team and several days collect multiple samples.
Shipping too is complicated by the use of organic solvent rinses and numerous
subsamples that require complex and expensive shipping.Sample analysis, after
collection, is also expensive. Multiple clean up steps are needed, and expensive
instrumentation is used, such as high resolution gas chromatography (HRGC)
coupled to high resolution mass spectrometry (HRMS). A low cost unitized
sampling system, the "AmbStack Sampler" was designed by our group,
and combined with a reporter gene bioassay system, the "CALUX"
method, to give accurate PCDD/PCDF analyses with much simpler techniques than
are currently in use. The AmbStack/CALUX system provides reliable air emission
data at a fractional cost of conventional emission methods.This system has been
demonstrated for ambient sampling, low temperature stack emissions, and simulated
industrial boiler discharges. The sampling unit, called the "AmbStack
Sampler”, is commercially available, and uses a polyurethane plug (PUF) insert
in a glass sampling cartridge for ambient and stack sampling. AmbStack samples
can be directly analyzed for PCDD, PCDF, PCB or polycyclic aromatic
hydrocarbons (PAH). The AmbStack Sampler contains a glass probe-cartridge unit,
and Teflon® connections a dry gas meter and air pump. After sampling, the
probe-cartridge unit is sealed with Teflon®-lined end caps, and shipped
directly to the laboratory for analysis. Sample extraction is done by in
situ solvent extraction and clean up, followed by CALUX reporter gene
bioassay.
Xenobiotic
Detection Systems, Inc. (XDS) has a genetically engineered cell line which
contains the firefly luciferase gene under trans-activational control of the
aryl hydrocarbon receptor. This cell line can be used for the detection and
quantification of AhR agonists. Exposure of this patented cell line to AmbStack
sample extracts yields a direct measure of total TEQ since response of these cells
is based on the mechanistic basis by which biologically active PCDD, PCDF, and
PCB express their toxicity (1,2).
In the current
experiments we demonstrate the sensitivity and performance of the AmbStack Sampler
for PCDD/PCDF quantification on a TEQ basis, using the CALUX bioassay and a simulated
industrial boiler discharge.
Materials and
Methods
Incinerator
Conditions
The combustion
system used to perform this test was a North American Package Boiler (NAPB), which
is capable of firing natural gas or #2 through #6 fuel oils. The boiler is a
three pass firetube “Scotch” marine-type design fitted with a North American
burner rated at 2.5 x106 Btu/hr. A dopant (a mixture of 1,2 dichlorobenzene and
copper naphthenate) was injected through a separate injection system to the
main fuel injection system prior to the burner. The dopant flow rate was adjusted
to yield a HCl concentration at the stack of approximately 500 ppm at 7% O2. A
Method 23 sampling train and the AmbStack sampler were placed at the same
location in the stack. The flue gas stream for this experiment was stable at a
temperature of about 140 oC with a mo isture level of about 11 %. Prior to
testing the Boiler unit experienced a thermal decontamination process of about
400 hours.
AmbStack
sampling and CALUX bioassay
The PUF insert
was removed from the cartridge, and the flow direction noted. The forward or
"front end" section of the PUF cartridge was separated from the
remaining PUF and analyzed separately with the probe rinse, to determine an
Apparent Collection Efficiency, ACE. The front 2/3 of the PUF insert was
extracted using toluene , and combined with the toluene rinsate from the probe
and the cartridge holder. The remaining back 1/3 section of the PUF insert was
extracted separately.The front and back extracts were analyzed separately to
determine if any sample breakthrough had occurred.
Sample extracts
were split into equal aliquots , the first aliquot was prepared by our Method 1
cleanup procedure to measure TEQ activity of chlorinated species (PCDD, PCDF,
and PCB). The second aliquot was prepared using our Method 2 Procedure which
provides separate extracts to estimate TEQ for PCB and PCDD/PCDF individually.
These proprietary clean up processes involve differential chromatography. All
extracts were solvent exchanged into DMSO before analysis.
Sample extracts
were suspended in cell culture medium. This media was applied to H1.1C2 mouse hepatoma
cells (Patent # 5,854,010 ) grown in 96 well culture plates. In addition to
sample dilutions a standard curve of 2,3,7,8-tectrachloro dibenzo-p-dioxin
(TCDD) was assayed. All assays of standards and unknowns were run in duplicate.
Plates were incubated for 4 hours in a humidified C02 incubator. Following
incubation media was removed and cells observed microscopically for viability.
Luciferase response, the induction of luciferase activity, was measured
optically as total light emission using a BMG Luminometer.
Results
Cell viability:
Microscopic examination of the cells following exposure to sample extracts did
not reveal any indication of toxicity. Samples were analyzed and compared to a
clean PUF blank.
Results of
CALUX measurements of TEQ activity from the simulated industrial boiler
extracts are presented in Table I.
TABLE I. CALUX
RESULTS (NANOGRAM TEQ ACTIVITY PER SAMPLE)
Method 1
|
Total
TEQ(PCDD/PCDF/PCB)A
|
|
Front End
(2/3 PUF/Rinsate)
|
13.7 ± 3.6
|
|
Back End (1/3
PUF)
|
2.8 ± 0.6
|
|
|
PCDD/PCDFB
|
PCBC
|
SumD
|
Method 2
|
TEQ
|
TEQ
|
Total TEQs
|
Front End
|
12.1 ± 2.07
|
2.01 ± 0.44
|
14.1
|
Back End
|
2.2 ± 0.47
|
0.91 ± 0.11
|
3.1
|
|
|
|
|
___________________________________________________________________
A. Data are
Mean ± Standard Deviation of 5 independent determinations in the CALUX assay
for total TEQ activity.
B. Data are
Mean ± Standard Deviation of 3 independent determinations in the CALUX assay
for TEQ activity in a sample fraction purified for dioxins and furans.
C. Data are
Mean ± Standard Deviation of 3 independent determinations in the CALUX assay
for TEQ activity purified for planar PCB.
D. Data are the
sum of TEQ determinations from PCDD/PCDF and PCB fractions.
Relative
emission levels found in collected PUF samples are presented in Table II based
on 3.58 M
3 air sampled
during the 3 hour test period.
TABLE II.
ANALYSIS OF AIR SAMPLES (NANOGRAM /METER3 TEQ ACTIVITY)
Method 1
|
Total TEQs
|
Front End
|
3.8
|
Back End
|
0.78
|
Apparent
Collection Efficiency
|
83%
|
|
|
Method 2
|
TEQ TEQ Total TEQ
|
|
PCDD/PCDF PCB
Sum
|
Front End
|
3.4 0.56 3.9
|
|
|
A comparison
HRMS analysis was performed by collecting a parallel U.S. Environmental
Protection Agency, Method 23 sample. The results of that analysis were 2.75
ng/dscm (7% O2) versus 1.9 ng/dscm (7% O2) by AmbStack and CALUX. The
comparison analysis of AmbStack/CALUX showed
excellent agreement with HRMS.
Discussion
The comparison
analysis of AmbStack/CALUX showed excellent agreement with HRMS. The AmbStack
Sampler, combined with CALUX TEQ quantification, gives a rapid and cost
effective method to measure PCDD/PCDF emissions. The method was sensitive at
concentrations found in a simulated industrial boiler emission. The CALUX
screen proved to be rugged in analyzing this complex sample type. The clean up
procedure was rapid and data reports were generated in two working days after
sampling was complete.
Performance in
this study suggests that the AmbStack/CALUX system, using PUF, is suitable for many
ambient and industrial applications, such as post control emissions testing.
This technique is especially useful as a low cost diagnostic tool to quickly
measure dioxin emissions from thermal combustion systems.
References
1) Garrison,
P.M., et al. Fund. Appl. Toxicol. 1996. 30, 194-203.
2) Denison,
M.S., A. Brouwer, and G.C. Clark. U.S. patent # 5,854,010.
Acknowledgement
The authors
would like thank Dr. Brian K. Gullet of the U.S. Environmental Protection
Agency for
his assistance
in arranging the boiler test, and facilitating sampling at the EPA Combustion
Research Facility at Research Triangle Park, NC.
Japanese analysis for ash and soil using CALUX
Full paper at:
http://www.dioxins.com/pdf/environment/environment10.pdf
Validation study for the use of the dioxin responsive CALUX assay for analysis of Japanese ash and soil samples
Brown D; Kishimoto Y; Ikeno O; Chu M; Nomura J; Murakami T; Murata H
Organohalogen Compounds 45:200, 2000
In Japan incineration is a common method for disposing of municipal waste and it is estimated that more than 10,000 incinerators of various capacities are currently in operation. In the past couple of years there has been an increased concern regarding the emission from these incinerators and other the emissions of other industries. In particular the concern has focused on the inadvertent production and release of chlorinate aromatic compounds such as polychlorinated biphenyls (PCBs), polychlorinated dibenzo--p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). In August 1997 the Japanese government addressed these concerns by amending the cabinet orders of the Air Pollution Control Law and Waste Management and PubIic Cleansing Law. These amendments implemented stricter regulations on incinerators and other Industries that emit PCBs, PCDDs and PCDFs. These amendments pIaced an immediate limit on emissions from new facilities that will emit these compounds and provided for gradually more strict limits for existing facilities over a five-year period. In order to comply with these new limits it was expected that monitoring by chemical analysis would have to increase. This raised concerns that the chemical analysis by HRGCMS for compliance might be an economic hardship for some of the regulated industries and that the increased demand might outstrip the capacity of the existing analytical laboratories. Based on these concerns the Japanese government and private corporations began to examine the possibility of using alternative testing methods to monitor for the presence of these compounds. In this article we report the results from a preliminary validation study conducted by Hiyoshi Corporation and Xenobiotic Detection Systems, Inc (XDS). This study used a blinded format to compare the results from the dioxin responsive CALUX assay with HRGCMS data.
The samples were extracted using a modification of the EPA 8290 extraction method
Briefly, the dried samples were ground and one gram aliquots were placed in solvent cleaned glass vials with PTFE lined caps. The sample was extracted with a 20% solution of methanol in toluene then twice with toluene. During each extraction step the samples were incubated in an ultrasonic water bath. The three extracts from each sample were filtered, pooled and concentrated by vacuum centrifugation. The sample extract was suspended in hexane and prepared for the bioassay by a proprietary clean up method. The eluate from the clean up method was concentrated under vacuum into dimethyl sulfoxide (DMSO). The DMSO solution was used to dose the genetically engineered cells in the CALUX assay. Prior to dosing the cells, the sample extracts in DMSO were suspended in cell culture medium. This medium was then used to expose monolayers of the H1L1 cell line grown in 96 well culture plates. In addition to the samples, a standard curve of 2,3,7,8-tetrachlorodibenzo-p-dioxin )TCDD) was assayed (161, 80.5, 40.2, 20.1, 10.1, 5.0, 2.5, 1.2 and 0.6 parts per trillion (ppt( TCDD). The plates were incubated for a time to produce optimal expression of the luciferase activity in a humidified CO2 incubator. Following incubation, the medium was removed and the cells were examined microscopically for viability. The induction of luciferase activity was quantified using the luciferase assay kit from Promega.
Results and Discussion
From the GC/MS analysis of the samples, the I-TEQs were calculated using the TEF values for the individual congeners. The sample I-TEQs were estimated by the CALUX assay by comparing the response of the sample extract to the standard curve for 2,3,7,8-TCDD. The correlation coefficient between the results is acceptable, (r = 0.94).
How the Japanese do an air sample
Sampling
Sample
should be taken by a high volume air sampler with which a sampling tube with 2 pieces
of polyurethane foam is attached below filter paper. For obtaining a 24-hour average
concentration, sample should be collected at a high flow rate of 700 L/min for
24 hours. For obtaining a weekly average
concentration, samples should be collected 7 times at a high flow rate of 700
L/min for 24 hours or collected continuously at a medium flow rate of 100
L/min for consecutive 7 days.
Glass fiber filter shall be used as the filter paper for a high-volume
air sampler.
Solvent Extraction
Sample is extracted from glass fiber filter by Soxhlet extractor
with toluene for 16 to 24 hours.
For the polyurethane foam, sample is extracted by a Soxhlet
extractor with acetone for 16 to 24 hours.
|
