Intra‐ and interlaboratory calibration of the DR CALUX® bioassay for the analysis of dioxins and dioxin‐like chemicals in sediments

Abstract

In the Fourth National Policy Document on Water Management in the Netherlands [1], it is defined that in 2003, in addition to the assessment of chemical substances, special guidelines for the assessment of dredged material should be recorded. The assessment of dredged material is based on integrated chemical and biological effect measurements. Among others, the DR CALUX® (dioxin responsive—chemically activated luciferase expression) bioassay has tentatively been recommended for inclusion in the dredged material assessment. To ensure the reliability of this bioassay, an intra- and interlaboratory validation study, or ring test, was performed, organized by the Dutch National Institute for Coastal and Marine Management (RIKZ) in cooperation with BioDetection Systems BV (BDS). The intralaboratory repeatability and reproducibility and the limit of detection (LOD) and quantification (LOQ) of the DR CALUX bioassay were determined by analyzing sediment extracts and dimethyl sulfoxide (DMSO) blanks. The highest observed repeatability was found to be 24.1%, whereas the highest observed reproducibility was calculated to be 19.9%. Based on the obtained results, the LOD and LOQ to be applied for the bioassay are 0.3 and 1.0 pM, respectively. The interlaboratory calibration study was divided into three phases, starting with analyzing pure chemicals. During the second phase, sediment extracts were analyzed, whereas in the third phase, whole sediments had to be extracted, cleaned, and analyzed. The average interlaboratory repeatability increased from 14.6% for the analysis of pure compound to 26.1% for the analysis of whole matrix. A similar increase in reproducibility with increasing complexity of handlings was observed with the interlaboratory reproducibility of 6.5% for pure compound and 27.9% for whole matrix. The results of this study are intended as a starting point for implementing the integrated chemical—biological assessment strategy and for systematic monitoring of dredged materials and related materials in the coming years.

Full text

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Environmental Toxicology and Chemistry, Vol. 23, No. 12, pp. 2781–2789, 2004
? 2004 SETAC
Printed in the USA
0730-7268/04 $12.00 ? .00
Environmental Chemistry
INTRA- AND INTERLABORATORY CALIBRATION OF THE DR CALUX? BIOASSAY
FOR THE ANALYSIS OF DIOXINS AND DIOXIN-LIKE CHEMICALS IN SEDIMENTS
HARRIE T. BESSELINK,*† COR SCHIPPER,‡ HANS KLAMER,‡ PIM LEONARDS,§ HENK VERHAAR,?? EMIEL FELZEL,†
ALBERTINKA J. MURK,# JOHN THAIN,†† KAZUNORI HOSOE,‡‡ GREET SCHOETERS,§§ JULIETTE LEGLER,???? and
BRAM BROUWER????
†BioDetection Systems B.V. (BDS), Badhuisweg 3, 1031 CM Amsterdam, The Netherlands
‡National Institute for Coastal and Marine Management/RIKZ, P.O. Box 8039, 4330 EA Middelburg, The Netherlands
§Netherlands Institute for Fisheries Research, P.O. Box 68, 1970 AB IJmuiden, The Netherlands
??OpdenKamp-Registration and Notification, Koninginnengracht 23, 2514 AB The Hague, The Netherlands
#Wageningen University, Subdepartment of Toxicology, P.O. Box 8000, 6700 EA Wageningen, The Netherlands
††Centre for Environment Fisheries and Aquaculture Science, Renbrance Avenue, CM0 8HA Burnham-on-Crouch, United Kingdom
‡‡Life Science Research Laboratories, Kaneka Corporation, Takasago, Hyogo 676-8688, Japan
§§Flemish Institute for Technological Research, Boeretang 200, B-2400 Mol, Belgium
????Institute for Environmental Studies, Vrije Universiteit Amsterdam, de Boelelaan 1115, 1081 HV Amsterdam, The Netherlands
(Received 3 October 2003; Accepted 30 April 2004)
Abstract—In the Fourth National Policy Document on Water Management in the Netherlands [1], it is defined that in 2003, in
addition to the assessment of chemical substances, special guidelines for the assessment of dredged material should be recorded.
The assessment of dredged material is based on integrated chemical and biological effect measurements. Among others, the DR
CALUX? (dioxin responsive–chemically activated luciferase expression) bioassay has tentatively been recommended for inclusion
in the dredged material assessment. To ensure the reliability of this bioassay, an intra- and interlaboratory validation study, or ring
test, was performed, organized by the Dutch National Institute for Coastal and Marine Management (RIKZ) in cooperation with
BioDetection Systems BV (BDS). The intralaboratory repeatability and reproducibility and the limit of detection (LOD) and
quantification (LOQ) of the DR CALUX bioassay were determined by analyzing sediment extracts and dimethyl sulfoxide (DMSO)
blanks. The highest observed repeatability was found to be 24.1%, whereas the highest observed reproducibility was calculated to
be 19.9%. Based on the obtained results, the LOD and LOQ to be applied for the bioassay are 0.3 and 1.0 pM, respectively. The
interlaboratory calibration study was divided into three phases, starting with analyzing pure chemicals. During the second phase,
sediment extracts were analyzed, whereas in the third phase, whole sediments had to be extracted, cleaned, and analyzed. The
average interlaboratory repeatability increased from 14.6% for the analysis of pure compound to 26.1% for the analysis of whole
matrix. A similar increase in reproducibility with increasing complexity of handlings was observed with the interlaboratory repro-
ducibility of 6.5% for pure compound and 27.9% for whole matrix. The results of this study are intended as a starting point for
implementing the integrated chemical–biological assessment strategy and for systematic monitoring of dredged materials and related
materials in the coming years.
Keywords—CALUX?
Dredged materialsDioxins Interlaboratory calibrationScreening bioassay
INTRODUCTION
It is generally accepted that marine sediments form a sink
for hydrophobic pollutants such as polychlorinated dibenzo-
p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs),
and polychlorinated biphenyls (PCBs). Since these classes of
compounds tend to bioaccumulate in aquatic organisms in-
habiting such polluted areas, they may pose a health risk for
aquatic wildlife and also for humans through the consumption
of contaminated fish. The occurrence of certain diseases in fish
populations has been related to environmental pollutants. Pol-
lution of the aquatic environment has been assumed to con-
tribute (at least in part) to the etiology of fish diseases such
as skin and liver tumors and fin rot [2–4]. The main source
of contamination in the North Sea is through riverine outflows,
primarily from the Rhine, Scheldt, and Meuse (The Nether-
lands), Elbe and Weser (Germany), and Thames (United King-
dom). As a consequence, coastal areas and estuaries are sig-
nificantly polluted by PCDDs, PCBs, and other persistent or-
* To whom correspondence may be addressed
(harrie.besselink@bds.nl).
ganic pollutants [5,6]. In order to monitor the extent of con-
tamination, the Dutch government has stated that the
assessment of dredged materials will be based on integrated
chemical and biological effect measurements [1,7]. The DR
CALUX? (dioxin responsive–chemically activated luciferase
expression) bioassay has been recommended for inclusion in
the dredged material assessment for the analysis of dioxins
and/or dioxin-like chemicals.
Traditional techniques for the detection and quantitation of
PCDDs, PCDFs, and PCBs in sediments involve costly and
time-consuming instrumental methods, such as high-resolution
gas chromatography separation and mass spectrometry
(HRGC/MS), making extensive monitoring of sediments dif-
ficult. Although these techniques provide information on the
presence and concentration of individual congeners, no direct
information is provided on the total biological (toxic) activity
of such compounds in complex mixtures. The major advantage
of using mechanistic-based bioassays for the assessment of
dredged materials, such as the DR CALUX bioassay, is that
instead of analyzing specific individual congeners, it deter-
mines the total biological (toxic) activity of groups of chem-

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Environ. Toxicol. Chem. 23, 2004H.T. Besselink et al.
icals with a similar toxic mode of action [7]. However, tra-
ditional analytical chemical techniques such as HRGC/MS are
essential to determine the exact nature of individual congeners
present in the samples under investigation.
The DR CALUX bioassay comprises a genetically modified
H4IIE rat hepatoma cell line, incorporating the firefly lucif-
erase gene coupled to dioxin responsive elements (DREs) as
a reporter gene for the presence of dioxins and/or dioxin-like
compounds [8–11]. In the DR CALUX bioassay, the induction
of luciferase by 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-
TCDD) is dose dependent. Hence, a 2,3,7,8-TCDD calibration
curve can be used to quantify the total dioxin and/or dioxin-
like content of a sample under investigation. Whereas con-
centrations of individual polyhalogenated aromatic hydrocar-
bons (PHAHs) as determined using HRGCMS have to be mul-
tiplied by their respective toxic equivalent factor (TEF) value
and added up to give the total 2,3,7,8-TCDD toxic equivalent
(TEQ) [12–14], the DR CALUX bioassay directly measures
the arylhydrocarbon receptor (AhR)–related toxic potency of
a mixture. The DR CALUX bioassay has been successfully
used for the analysis of dioxins and/or dioxin-like chemicals
in a wide variety of matrices, such as (human) serum, (human)
milk, fish, fish oil, citrus pulp, and so on [15–20]. In addition,
a number of papers have been published describing the vali-
dation of the bioassay and describing the correlation between
DR CALUX and HRGCMS derived 2,3,7,8-TCDD TEQs [21–
24].
To ensure the reliability of the DR CALUX bioassay for
monitoring dredged materials, the accuracy and repeatability
of the DR CALUX bioassay has to be determined. Therefore,
both intra- and interlaboratory calibration studies were per-
formed. Six laboratories, located in The Netherlands, the Unit-
ed Kingdom, Japan, and Belgium, were selected to participate.
Each of these laboratories analyzed anonymous sediment sam-
ples in each of the three phases of the study. The participants
were asked to perform the analyses according to supplied pro-
tocols. In addition, they were asked to extract and clean up
sediments according to the procedure developed and validated
by The Netherlands Institute for Fisheries Research (RIVO),
in cooperation with the Dutch National Institute for Coastal
and Marine Management. The protocol for the analysis of
dioxin and/or dioxin-like content in sediment extracts (DR
CALUX bioassay) was recently modified by BioDetectionSys-
tems BV (BDS) (SOP Dutch National Institute for Coastal
Marine Management SPECIE*07). OpdenKamp performedthe
statistical analyses necessary to interpret the results of this
interlaboratory validation study. The results of these studies
are presented here and are intended as a starting point for
implementation of the DR CALUX bioassay in the assessment
of dredged materials for systematic monitoring in the coming
years.
MATERIALS AND METHODS
Chemicals
The 2,3,7,8-TCDD was purchased from LGC Promochem
(Wesel, Germany). The PCB 126 and PCB 169 were obtained
from Labor Dr. Ehrenstorfer (Augsburg, Germany). Ultraclean
DMSO and ethylenediaminetetraacetic acid (EDTA) were ob-
tained from Acros (New Brunswick, NJ, USA). Ultra resi-
analyzed n-hexane, sodium sulfate, and cleaned and ignited
sea sand were from Mallinckrodt Baker B.V. (Deventer, The
Netherlands). Silica 60 (63–200 ?m) was obtained from Merck
(Darmstadt, Germany). Sulfuric acid (95–97%) was from Rie-
del de Hae ¨n (Seelze, Germany). Minimal essential medium (?-
MEM with phenol red as pH indicator), fetal calf serum (Aus-
tralian origin), and trypsin were purchased from Gibco, In-
vitrogen (Breda, The Netherlands). Dithiothreitol (DTT) and
Luciferin was from Duchefa Biochemie B.V. (Haarlem, The
Netherlands). Coenzyme A (free acid grade I) was from Roche
Diagnostics (Mannheim, Germany).
Intralaboratory study
The intralaboratory calibration study was performed by the
Institute for Environmental Studies. Sediment was extracted
and cleaned up as indicated here. The determination of dioxin
and/or dioxin-like content was according to the method in-
dicated under the section DR CALUX analysis. For the in-
tralaboratory study, the following parameters were investi-
gated: limit of detection (LOD), limit of quantitation (LOQ),
and reproducibility and repeatability of the bioassay.
Interlaboratory study
Project description. Six laboratories, located in The Neth-
erlands, the United Kingdom, Japan, and Belgium, were se-
lected to participate. Each of these laboratories analyzed blind
samples in each of three phases of the study. The participants
were asked to perform the analyses according to supplied pro-
tocols (see the following discussion). All samples supplied
were analyzed three times. In addition, each single sample was
analyzed in triplicate. All the laboratories participating in the
interlaboratory study for the validation of the DR CALUX
bioassay for dioxins and dioxin-like chemicals in sediment
had experience in running the DR CALUX bioassay. However,
none of the participating laboratories had prior experience with
the extraction protocol to be used. Furthermore, the partici-
pants were free to use the dilution factor of their choice unless
indicated otherwise.
Phase 1. The first phase of the interlaboratory study con-
sisted of the DR CALUX analysis of two defined standard
solutions (2,3,7,8-TCDD in DMSO; TCDD/PCB-126/PCB-
169 mix in DMSO). The standard solutions were prepared by
the Institute for Environmental Studies, Vrije Universiteit Am-
sterdam, The Netherlands, and sent to the participants. In ad-
dition, the participants received a complete concentration
range of 2,3,7,8-TCDD in DMSO to be used as a TCDD cal-
ibration curve (a total of eight different TCDD concentrations,
0–300 pM 2,3,7,8-TCDD/well, including DMSO as a blank
control). This calibration curve was used throughout the whole
interlaboratory study. Furthermore, each participant analyzed
its own TCDD calibration curve. Participating laboratories re-
ceived three vials containing a TCDD stock solution in DMSO
and three vials containing a TCDD/PCB-126/PCB-169 mix in
DMSO. Dilutions of the stock solutions were prepared by the
participants in DMSO and tested for dioxin and/or dioxin-like
content. Raw data as well as converted data were used for
statistical evaluation.
Phase 2. In the second phase of the study, the participants
were asked to analyze three extracted and cleaned sediment
samples using the DR CALUX bioassay. Sediments used for
extraction and cleanup were freshwater sediments from the
Western Scheldt, The Netherlands. The sediment extracts were
prepared by the Royal Institute for Fishery Research (RIVO-
DLO), IJmuiden, The Netherlands, according to the protocol
given here. Dilutions of the supplied sediment extracts were
prepared by the participants in DMSO and tested for dioxin
and/or dioxin-like content. On each 96-well microtiter plate,

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Interlaboratory calibration of the DR CALUX? bioassay
Environ. Toxicol. Chem. 23, 20042783
a 2,3,7,8-TCDD standard calibration curve was analyzed. Raw
data as well as converted data were used for statistical eval-
uation.
Phase 3. During phase 3 of the interlaboratory study, par-
ticipants received an identical sediment sample (freshwater
sediment from the Western Scheldt, The Netherlands). The
participants were asked to extract and clean up the sediment
in three separate sessions according to the supplied protocol.
Following extraction and cleanup, the three sediment extracts
were analyzed in the DR CALUX bioassay. Participants were
not instructed on the dilution to be used. Dilutions of the
sediment extracts were prepared by the participants in DMSO
and tested for dioxin and/or dioxin-like content. On each 96-
well microtiter plate, a 2,3,7,8-TCDD standard calibration
curve was analyzed. Furthermore, an appropriate commer-
cially available procedure blank (washed and ignited sea sand;
Baker, catalog 0252) was extracted, cleaned, and analyzed us-
ing the exact same protocols. Raw data as well as converted
data were used for statistical evaluation.
Preparation of samples for the intra- and
interlaboratory studies
Defined standard solutions. The two defined standard so-
lutions were prepared by dissolving either 2,3,7,8-TCDD or
2,3,7,8-TCDD, PCB 126, and PCB 169 in DMSO. The 2,3,7,8-
TCDD standard solution contained 2,3,7,8-TCDD in DMSO
at a concentration of 7.5 nM. Sample 2 contained a mixture
of 2,3,7,8 TCDD, PCB 126, and PCB 169 at concentrations
of 5.0, 25, and 250 nM, respectively. The total 2,3,7,8-TCDD
TEQ content of this mixture was calculated using both World
Health Organization (Paris, France) WHO-TEF values and DR
CALUX-relative potency (REP) values [25] and found to be
10 and 7.5 nM 2,3,7,8-TCDD TEQ, respectively. Overall, 27
individual measurements per sample and per participant were
available for data analysis.
Extraction of sediment samples. Prior to extraction, sedi-
ment samples were freeze-dried and homogenized. Approxi-
mately 10 g of dried sediment were placed in a preextracted
thimble, and a small piece of silanized glass wool was placed
in the thimble on top of the sample to prevent sediment parts
from leaving the thimble. The thimble was placed in a Soxhlet
setup and extract for 16 h (overnight) with 200 ml hexane/
acetone (3/1 v/v). The extracts were concentrated in the ro-
tation evaporator until approximately 5 ml (p ? 0.05 bar;
T ? 40?C) of extract remained. If the extract still contained
solid particles, the extract was filtered with diatomaceous earth
or sodium sulfate. The extract was transferred to a diatoma-
ceous earth– or sodium sulfate–filled funnel and flushed with
10 ml hexane. The eluted extract was evaporated again in the
rotary evaporator until approximately 5 ml of extractremained.
The extract was transferred to a cleaned glass tube and con-
centrated until near dryness. The dried extract was finally re-
dissolved in 3 ml hexane.
Cleanup of sediment samples. The extracted sediment sam-
ples were cleaned up using a multilayer column. Themultilayer
glass column consisted of the following materials (from top
to bottom): 1 cm water-free sodium sulfate, 1 g silica, 7 g 44%
sulfuric acid on silica, 1 g silica, 2 g 33% sodium hydroxide
on silica, 1 g silica, 1.5 g 10% silver nitrate on silica, and a
small piece of silanized glass wool. After addition of each
layer, the column was compacted by tapping the column. After
moistening and preelution with 25 ml of hexane of the column,
the complete extract was transferred on the top of the column.
The column was eluted with 130 ml of hexane, after which
the eluate was concentrated on the rotation evaporator until
approximately 5 ml remained (p ? 0.2 bar; T ? 40?C). The
concentrated cleaned sediment extract was transferred to a
clean glass tube and further concentrated to near dryness under
a gentle stream of nitrogen. The extract was redissolved in 50
?l DMSO.
DR CALUX analysis
The DR CALUX cells were cultivated in minimal essential
medium (?-MEM) supplemented with 10% fetal calf serum
under standardized conditions (37?C, 5% CO2, 100% humid-
ity). The DR CALUX analyses of samples in DMSO of the
three phases were performed in 96-well cell culture plates
(Greiner). Cells were seeded in 100 ?l growth medium and
incubated for 24 h under standardized conditions until the cells
reached a confluence of at least 95%. An additional 100 ?l of
growth medium were added to the wells containing the samples
in DMSO. The final DMSO concentration in the wells was
0.4%. After 24 h of incubation, the exposure medium was
removed, and the cells were rinsed with diluted phosphate
buffered saline (demi water/[PBS]; 1/1, v/v). Thirty microliters
of lysis mix (25 mM Tris, 2 mM dithiothreitol [DTT], 2 mM
1,2-diaminocyclohexane-N,N,N?,N-tetra-acetic acid [CDTA],
10% glycerol, and 1% Triton x-100 [Sigma, St. Louis, MO,
USA] pH 7.8) were added to each well and incubated at 4?C
for at least 30 min, after which the microtiter plates werefrozen
at ?80?C for a minimum of 30 min and a maximum of 1 d
to lyse the cells.
The luciferase activity was measured using a luminometer
equipped with two dispensers. The microtiter plates were
thawed and shaken for 2 min at room temperature and placed
in the luminometer. One hundred microliters of glow mix (20
mM trycin, 1.07 mM magnesium hydroxide carbonate pen-
tahydrate, 2.67 mM magnesium sulfate, 0.1 mM ethylenedi-
aminetetraacetic acid, 33.3 mM dithiothreitol, 270 ?M co-
enzyme A, 470 ?M luciferin) were automatically injected into
each well. The light output was recorded on which the reaction
was stopped by automatic injection of 100 ?l of 0.2-M NaOH.
On each 96-well microtiter plate, a complete 2,3,7,8-TCDD
standard concentration range was incubated and analyzed in
triplicate. A curve fit of the 2,3,7,8-TCDD standard range was
produced for the calculation of DR CALUX TEQ content in
the samples tested. The analyzed relative light units (RLU)
from the samples were interpolated on the 2,3,7,8-TCDD stan-
dard curve, and the DR CALUX TEQ content was quantified
between the limit of quantitation (LOQ) and the concentration
of 2,3,7,8-TCDD at which 50% of the maximum response is
observed (EC50).
Statistical analysis
To maintain consistency of statistical analyses, an identical
microtiter plate setup was used by all participants, and all
samples were analyzed in an identical manner. Both raw data
and pretreated data from analyzed samples were submitted to
OpdenKamp Registration and Notification for statistical eval-
uation. Data pretreatment consisted of all necessary calcula-
tions to convert the luminosity readings as submitted by the
participating laboratories to effective dioxin-receptor activity
(pM 2,3,7,8-TCDD TEQ). In addition to the analysis results
of the defined samples (phase 1), the cleaned sediment extracts
(phase 2), and the complete sediments (phase 3), all partici-

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Environ. Toxicol. Chem. 23, 2004H.T. Besselink et al.
Fig. 1. Example of a 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-
TCDD) standard calibration curve. The relative light units have been
corrected for dimethyl sulfoxide blank. r2? 0.993.
Table 1. Intralaboratory repeatability and reproducibilityofthedioxin
response–chemically activated lucerferase (DR CALUX?) bioassay
for sediment extracts; 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-
TCDD); TEQ ? toxic equivalent
DR CALUX analysis result
(pg 2,3,7,8-TCDD TEQ/g sediment)
Repeatabilityab
Sediment 1Sediment 2
Reproducibilityab
3 pM 2,3,7,8-
TCDD Sediment 4
Average
SD
%SD
4.03
0.97
24.1
25.73
2.54
9.9
2.96
0.41
13.8
23.06
4.58
19.9
aFor the determination of the repeatability and reproducibility, each
sample/extract analysis was analyzed 10 times in triplicate.
bThe repeatability and reproducibility are calculated as percentage
standard deviation (%SD).
pants also submitted the results of the complete 2,3,7,8-TCDD
calibration curves for statistical evaluation.
Calibration curves were fitted, and EC50 values were de-
rived using the nonlinear regression package pro Fit 5.5
(QuantumSoft, Zu ¨rich, Switzerland). The results of the cali-
bration curve measurements were fitted to a sigmoidal dose–
response function of the following form with a slope factor
of 1:
R
? R
max0
R ? R ?
0
(EC50?LogC)
1 ? 10
with C ? concentration, R ? response, R0? control response,
Rmax? maximum response, and EC50 ? the concentration at
which 50% response is observed. The R0was fixed in all fits
to the response of the control sample at concentration 0; Rmax
and EC50 were fit by a nonlinear least-squares algorithm (the
default Levenberg–Marquardt algorithm).
Analysis of variance (ANOVA) analyses were performed
using the general statistical package StatView 5.01 (SAS In-
stitute, Cary, NC, USA). The ANOVAs were calculated as
repeated-measures ANOVAs with wells as within factor for
phase 1 and with plates as within factor for subsequent phases.
Specialized statistics, such as comparison of fits of different
calibration curves, were calculated in MATLAB 5.1
(MathWorks, Natick, MA, USA) using custom routines.
RESULTS
Intralaboratory study
For the determination of the limit of detection (LOD) and
LOQ, 10 standard 2,3,7,8-TCDD calibration series were an-
alyzed in triplicate using the DR CALUX bioassay. In Figure
1, a typical example of a standard 2,3,7,8-TCDD calibration
curve is given. For each individual calibration curve, the LOD
was calculated as three times the standard deviation of the
DMSO blank (0 pM 2,3,7,8-TCDD), whereas the LOQ was
calculated as 10 times the standard deviation of the DMSO
blank [26]. For the 10 standard 2,3,7,8-TCDD calibration
curves, the LOD varied between 0.04 and 0.25 pM 2,3,7,8-
TCDD per well. The LOQ varied between 0.12 and 0.88 pM
2,3,7,8-TCDD per well. Finally, an overall LOD and LOQ was
calculated as the average of 10 observations plus three times
the standard deviation (95% confidence) resulting in a LOD
and LOQ of 0.3 and 1 pM 2,3,7,8-TCDD per well, respectively.
For the determination of the repeatability, two sediments
originating from coastal areas along the Dutch coastline were
extracted and cleaned up. One of the sediments had a low
2,3,7,8-TCDD TEQ content, whereas the second sediment had
a relatively high 2,3,7,8-TCDD TEQ content (4.8 and 26 pg
2,3,7,8-TCDD TEQ/g sediment, respectively). The 2,3,7,8-
TCDD TEQ content in both extracts was determined by DR
CALUX analysis 10 times on the same day. Thereproducibility
was determined by analyzing a 3-pM 2,3,7,8-TCDD standard
and a cleaned sediment extract. Both samples were analyzed
on 10 different days and by various persons. The results are
summarized in Table 1.
The linearity of response of the bioassay depends on the
linearity of the luminometer used. To determine the linearity
of response, a concentration range of luciferase was prepared
and the activity measured. A good linear correlation between
the detected amount of light and the luciferase concentration
was found (r2? 0.9997).
Interlaboratory study
Phase 1: 2,3,7,8-TCDD calibration curves. In phase 1 of
the ring test, all six laboratories analyzed two calibration
curves per microtiter plate, a BDS-supplied calibration curve,
as well as a calibration curve prepared in house by the par-
ticipants themselves. In Table 2, the EC50 values and the co-
efficients of determination for the curve fits for all participants
are summarized. In addition, the 3-pM point of the 2,3,7,8-
TCDD calibration curve is given. Differences in EC50 values
reported by the participating laboratories are apparent. In par-
ticular, participant B reported relatively high EC50 values in
both the calibration series provided by the coordinator and the
calibration series prepared by participant B themselves.
Both the EC50 values and the 3-pM point of the 2,3,7,8-
TCDD calibration curve serve as quality criteria. For each
participant, the results for both data points from all 96-well
plates analyzed during the presented study were collected and
recorded in Shewhart control charts. The Shewhart control
chart is used to identify variations on performance of the DR
CALUX bioassay brought about by unexpected or unassigned
causes. The Shewhart control chart shows the mean of the
EC50 and 3-pM control point and the upper and lower control
limits. In Figure 2, a typical Shewhart control chart is shown.
Over the analysis period, none of the participants exceeded
the action levels (AVG ? 3·S).
The results of the multiple analysis of the standard 2,3,7,8-

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Interlaboratory calibration of the DR CALUX? bioassay
Environ. Toxicol. Chem. 23, 20042785
Table 2. Summary of dioxin responsive–chemically activated lucerferase (DR CALUX?) analysis result (pM 2,3,7,8-tetrachlorodibenzo-p-dioxin
[2,3,7,8-TCDD]/well) for the 2,3,7,8-TCDD calibration curves
Participant
Calibration curve provided by coordinator
EC50a
(pM TCDD/well)
r2
Calibration curve prepared by participant
EC50
(pM TCDD/well)
r2
3 pMb
(pM/well)
A
B
C
D
E
F
6.22
21.7
11.0
13.9
10.0
10.9
0.997
0.994
0.999
0.999
0.999
0.960
8.88
26.9
9.43
12.6
17.1
12.5
0.997
0.999
0.998
0.988
0.997
0.986
2.80
2.44
3.02
3.04
2.98
3.10
aMedian effective concentration.
bResult of the 3 pM 2,3,7,8-TCDD calibration concentration prepared by the participants.
Fig. 2. Typical example of a Shewart control chart of the 3-pM point
of the 2,3,7,8-tetrachlorodibenzo-p-dioxin(2,3,7,8-TCDD)calibration
curve.
Table 3. Averaged limits of detection (LOD) and limitsofquantitation
(LOQ) (pM 2,3,7,8-tetrachlorodibenzo-p-dioxin [2,3,7,8-TCDD]/
well) over all experiments, by participant; SD ? standard deviation;
%SD ? percentage standard deviation
Participant
LOD
AverageSD
LOQ
AverageSD
A
B
C
D
E
F
Average
SD
%SD
0.17
0.41
0.36
0.29
0.43
0.21
0.31
0.11
34.8
0.09
0.47
0.33
0.07
0.37
0.14
0.52
0.92
0.95
0.75
1.16
0.61
0.82
0.24
28.8
0.31
0.92
0.85
0.19
0.91
0.28
TCDD calibration curves are also be used to determine the
per-participant LOD and LOQ taking into account interlabor-
atory variation (Table 3). This results show that on average,
the participants of the calibration study meet the set LOD and
LOQ derived from the intralaboratory study.
Phase 1: Defined standard solutions. Participants were
asked to measure the response of the two standard samples in
the DR CALUX bioassay three times in triplicate. The total
DR CALUX 2,3,7,8-TCDD TEQ content of both the 2,3,7,8-
TCDD sample as well as the mixed sample was calculated to
be 7.5 nM TEQ. Since the DMSO content during exposure
was 0.4% and the samples were diluted seven times by all
participants, the expected DR CALUX TEQ content per well
for both samples was 4.3 pM 2,3,7,8-TCDD TEQ. Overall, 27
individual measurements per sample and per participant were
available for data analysis. Averaged results for the concen-
tration of dioxin equivalents per participant and per sample
are summarized in Table 4. The DR CALUX results for the
dioxin sample are slightly higher than the actual 2,3,7,8-TCDD
concentration in the sample. The results for the TCDD/PCB
mixed sample are on average lower than the 2,3,7,8-TCDD
TEQ content as calculated using CALUX REP values for the
individual congeners.
Phase 2: Sediment extracts. In phase 2, participants were
provided with three extracted sediment samples, all originating
from the same batch. The participants were advised to dilute
the supplied samples 10? and 30?. Since the three samples
were analyzed on three separate plates, nine measurement val-
ues per extract dilution were available per participant. The
ANOVA results for the sediment extract samples, when ana-
lyzed by individual participant, show that significant differ-
ences exist between the results obtained per laboratory (p ?
0.0001) and also between the two dilutions employed (p ?
0.0007). It was observed that DR CALUX analysis of the 30?
diluted samples give higher results than the 10? diluted sam-
ples (data not shown). Averaged results for the concentration
of 2,3,7,8-TCDD TEQs per participant and per sample (30?
diluted) are given in Table 4. Quantitatively Bonferroni–Dunn
multiple comparisons indicate that overall (taken over both
dilutions), participant C is significantly different from the rest.
Although not traceable anymore, and because the results ob-
tained from the three control samples (DMSO blank, 3-pM
2,3,7,8-TCDD control, and internal reference sample) com-
plied with the quality performance criteria for the DR CALUX
bioassay, the indication is strong that a dilution error was made
by participant C in the supplied sample.
In addition to the sediment extracts, all participantsreceived
a procedure blank. The procedure blank is analyzed to check
for possible contamination from chemicals and materials used
during extraction and/or cleanup. DR CALUX analysis results
from this procedure blank for all participants were below the
limit of quantitation (1 pM 2,3,7,8-TCDD TEQ/well) and
therefore comply with the DR CALUX performance criteria
(data not shown).
Phase 3: Sediment sample. In phase 3, participants were
provided with a single contaminated sediment. Theparticipants
were asked to extract and clean up the sediment in three sep-
arate sessions using the Soxhlet extraction method. Following
extraction and cleanup, the three sediment extracts were an-
alyzed using the DR CALUX bioassay method. Participants
were left free to choose their own dilutions. The different
dilutions chosen made it impossible to perform variance anal-
yses by participants ? dilutions. Analysis of variance by par-

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Environ. Toxicol. Chem. 23, 2004H.T. Besselink et al.
Table 4. Dioxin responsive–chemically activated lucerferase (DR CALUX?) repeatability and reproducibility for the bioanalysis of the defined standard solutions and sediment samples of the various
phases of the present interlaboratory validation study
Participant
Dioxin sample
TCDD TEQ
(pM)a
Repeatability
(%SD)b
Mixed sample
TCDD TEQ
(pM)a
Repeatability
(%SD)b
Sediment extract
TCDD TEQ
(pg/g)a
Repeatability
(%SD)b
Sediment
TCDD TEQ
(pg/g)a
Repeatability
(%SD)b
A
BC
D
E1c
4.5 4.5 5.14.64.5
9.4
21.0
8.4 1.0
17.0
3.73.2 4.54.34.2
12.1 13.911.3 10.515.5
41.538.826.5 38.125.5
17.1 19.4
8.86
8.6
19.5
5.2 2.85.1 4.73.1
8.3
20.6
5.8 5.8
37.8
E2c
F
Average repeatability (%SD) Average (pM)
Reproducibility (%SD)
4.8 4.24.66.5
11.434.2 14.6
4.24.04.0
10.5
12.9 35.9 16.0
35.9 33.8 34.318.0
3.1
28.4 15.0
4.4 2.64.0
27.9
47.656.826.1
aData are expressed either as pM 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) toxic equivalents (TEQ)/well or as pg 2,3,7,8-TCDD TEQ/g extracted sediment (dry wt).
bThe repeatability and reproducibility are calculated as percentage standard deviation (%SD).
cParticipant E performed all DR CALUX analyses twice. Here, both reported results are taken into account.
Fig. 3. Averaged dioxin responsive–chemically activated lucerferase
(DR CALUX?) results by participant for the sediment extracted by
the participants (phase 3). Participants were free to choose their own
dilution factor for analysis. Data presented are 0? to 200? diluted
samples; 2,3,7,8-TCDD ? 2,3,7,8-tetrachlorodibenzo-p-dioxin; TEQ
? toxic equivalents.
ticipants indicates that significant differences exist between
laboratories (data not shown). Figure 3 shows graphical rep-
resentations of the results by participants. The results are cat-
egorized as 0? to 200? dilutions. Since not all the participants
submitted results in higher dilution ranges, these results were
not evaluated. However, it should be noted that higher re-
sponses were observed by participants at higher dilutions as
compared to the 0 to 200 category as presented here. In the
lowest dilution category, significant differences (p ? 0.006)
exist between participants, mainly because of low analytical
responses for participant F. Averaged results for the concen-
tration of 2,3,7,8-TCDD TEQs per participant and persediment
are given in Table 4.
Repeatability and reproducibility. The repeatability of the
DR CALUX bioassay was calculated for all samples (2,3,7,8-
TCDD sample, mixed sample, sediment extract, and sediment
sample) analyzed by the participants over the three phases of
the interlaboratory validation study as the relative standard
deviations of the obtained results (Table 4). The average re-
peatability for the participating laboratories ranged from
14.6% for the dioxin sample analysis to 26.1% for the sediment
samples that had to be extracted by the participantsthemselves.
It can be seen that the repeatability was lowest for phase 3,
during which the participants were asked to extract, clean up,
and perform a DR CALUX bioassay on a supplied sediment
sample. In Table 4, the reproducibility for the various analyzed
samples is also given. The percentage standard deviations over
the DR CALUX bioanalysis results for the analyzed samples
ranged from 6.5% for the dioxin sample to 27.9% for the
supplied sediment sample. Again, the biggest differences in
analysis results were observed in the sediment sample that had
to be extracted and cleaned up by the participants themselves.
DISCUSSION
The aim of the present study was to identify theDRCALUX
bioassay performance criteria for the analysis of PHAHs in
sediment samples in order to implement the bioassay in the
assessment of dredged materials for systematic monitoring in
the coming years. Therefore, both an intra- and interlaboratory
validation study was performed.

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Interlaboratory calibration of the DR CALUX? bioassay
Environ. Toxicol. Chem. 23, 20042787
Standard 2,3,7,8-TCDD calibration curves
Calibration curves were made with a dioxin standard and
were used to convert DR CALUX response levels to concen-
trations expressed as 2,3,7,8-TCDD TEQs. The section of the
2,3,7,8-TCDD calibration curve between the LOQ (1 pM) and
the EC50 is used to quantify DR CALUX analysis results. This
section is not linear (see Fig. 1). However, when the calibration
curve is plotted on a linear–linear scale, the indicated region
can be regarded as linear. In addition, the region between the
LOQ and EC50 is chosen for quantification of analysis results
since this region of the 2,3,7,8-TCDD calibration curve is least
sensitive to variations in observed DR CALUX activity.
Because the 2,3,7,8-TCDD calibration curve is used for
quantification of analysis results, the stability and quality of
the calibration curves is important. Furthermore, the calibra-
tion curves themselves are used as a DR CALUX bioassay
quality criterion. According to the performance criteria set for
the DR CALUX bioassay, the fitted EC50 should be within
the range of 6 to 18 pM 2,3,7,8-TCDD; otherwise, the results
are rejected. In addition, the EC50 value of 2,3,7,8-TCDD
should be constant over a longer time period. Finally, the
coefficient of determination (r) should be more then 0.95 [27].
The numerical results of the fit are summarized in Table 2.
Differences in EC50 values between labs are apparent.
Since actual fits are based on log (concentration) values and
therefore yield log EC50 estimates, differences in these esti-
mates will be exaggerated when transforming these values to
EC50s. In addition, high EC50 values reported by participant
B are correlated to low relative responses at the low end of
the concentration range, up to and including 3.0 pM (individual
curve fits not shown). Based on the results of the present study,
EC50 values may range between 8.3 and 18.1 pM (based on
a relative error of 15.4%). In a number of previous studies,
EC50 values in the same range as suggested previously were
found [20,21,28–30]. For the moment, the EC50 value of the
2,3,7,8-TCDD calibration curve is used as a quality control
for the 2,3,7,8-TCDD calibration curve. It can be observed
that EC50 values may differ between persons performing the
DR CALUX bioassay but also between analyses performed by
a single person. However, fluctuating EC50 values do not in-
terfere with the final results of a DR CALUX analysis, es-
pecially with data quantified below the EC50 of the standard
curve. Observed differences in the 2,3,7,8-TCDD calibration
curves occur mainly at the high response end of the calibration
curves above the EC50. Since the high end of the calibration
curve is not used for data interpolation, differences do not
significantly influence analysis results. Most probably, the
EC50 value is an indication of the quality or condition of the
cells rather than a performance criterion. Despite differences
between individual calibration curves, the coefficient of de-
termination for the individual analyzed 2,3,7,8-TCDD cali-
brations curves is high, and the 3-pM 2,3,7,8-TCDD concen-
tration of the 2,3,7,8-TCDD calibration curves prepared by the
participants themselves showed good comparability between
the participating laboratories. The 3-pM 2,3,7,8-TCDD con-
centration is used as quality control and is registered on a
Shewhart control chart. The calculated average value was 2.91
pM (standard deviation ? 0.21). The percentage standard de-
viation was calculated to be 7.2%.
WHO-TEFs versus DR CALUX-REPs
The WHO-TEF values are internationally accepted toxic
equivalent factors for dioxins, furans, and dioxin-like PCBs,
as stated by the WHO and derived from both in vivo and in
vitro studies. The relative toxic potency of dioxins, furans, and
dioxin-like PCBs, relative to 2,3,7,8-tetrachlorodibenzo-p-di-
oxin (2,3,7,8-TCDD), can also be derived from analyzing the
response elicited by various congeners using the DR CALUX
bioassay. The potencies found using this method are expressed
as CALUX REPs (CALUX relative potencies). The CALUX
REP values are actual TEF values for the congeners in the DR
CALUX bioassay and represent the total toxic potency of all
congeners present that show affinity toward the Ah receptor.
A number of authors have compared WHO TEFS and DR
CALUX REPs [25,28,29]. Differences between WHO TEFs
and DR CALUX REPs are apparent. As a consequence, DR
CALUX TEQs differ from HRGCMS-analyzed 2,3,7,8-TCDD
TEQs in a given sample because of the difference between
WHO TEFS and DR CALUX REPs [24,31]. This was dem-
onstrated for the mixed sample from phase 1 analyzed by the
participants. Furthermore, differences in DR CALUX–derived
TEQs and HRGCMS-derived TEQs can be a result of the fact
that by using HRGCMS, only specified dioxin and/or dioxin-
like compounds are determined. In contrast, all compounds
showing affinity toward the Ah receptor are detected by the
DR CALUX bioassay.
LOD and LOQ
The LOD and the LOQ of the DR CALUX bioassay were
determined by analyzing 10 standard 2,3,7,8-TCDD calibra-
tion series. From these analyses, it was concluded that taking
into account 95% confidence, a LOD and LOQ of 0.3 and 1
pM 2,3,7,8-TCDD per well, respectively, should be applied.
Hence, in case 10 g of sediment are processed and analyzed
using 0.4% of DMSO per well, the LOD and LOQ can be
calculated to be 0.04 and 0.16 pg 2,3,7,8-TCDD equivalents
per gram of sediment. Similar LOD and LOQ were reported
by a number of authors [17,30].
The participants of the interlaboratory validation study also
analyzed multiple standard 2,3,7,8-TCDD calibration curves.
From these data, a per-participant LOD and LOQ could be
determined. On average, the participants of the calibration
study met the set LOD and LOQ derived from the intralabor-
atory study. Furthermore, analysis of variance indicated that
no significant differences in LOD between laboratories could
be identified.
Effect of dilutions
The sediment extracts were analyzed at 10? and 30? di-
lutions. Whereas the 10? diluted samples showed an average
DR CALUX TEQ content over all participants of 27.3 ? 4.0
pg 2,3,7,8-TCDD TEQ/g sediment, the 30? diluted extract
gave a DR CALUX response of 34.9 ? 6.1 pg 2,3,7,8-TCDD
TEQ/g sediment. In general, an effect of dilution on the total
DR CALUX TEQ content in sediment samples is observed.
Although the exact nature for this observation is not known,
it is hypothesized that this is due to the presence of various
compounds in sediment extracts showing variable affinity to-
ward the Ah receptor. Dose–response curves in the DR CA-
LUX bioassay of individual compounds have been studied and
showed obvious differences [25] both in maximum response
and slope of the curve fit.
Repeatability
For the determination of the intralaboratory repeatability
of the DR CALUX bioassay for sediment samples, two sed-

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Environ. Toxicol. Chem. 23, 2004H.T. Besselink et al.
iment extracts were analyzed 10 times. Each analysis was per-
formed in triplicate. As a prerequisite for a correct triplicate
analysis, the percentage standard deviation in the triplicate
determination should be below 15%. This is in accordance
with the harmonized quality criteria for cell-based bioassay
analyses of PCDDs/PCDFs in feed and food as formulated by
Behnisch et al. [27] and as detailed in European Union direc-
tive 2002/69/EC and directive 2002/70/EC. The repeatability
for the low-2,3,7,8-TCDD-content sediment extract was found
to be 24.1% whereas in the high-content-sediment extract, the
repeatability was shown to be 9.9%.
For each participating laboratory, the repeatability of the
DR CALUX bioassay was calculated for the four samples
(2,3,7,8-TCDD sample, mixed sample, sediment extract, and
sediment sample) analyzed by the participants over the three
phases of the interlaboratory validation study. The average
repeatability for the participating laboratories ranged from
14.6% for the dioxin sample analysis to 26.1% for the sediment
samples that had to be extracted by the participants. In an
interlaboratory comparison exercise for the analysis of PCDD/
PCDFs in digested sewage sludge using HRGCMS, relative
standard deviations for standard solutions varied between 15
and 41% [32]. Similar ranges of relative standard deviations
were reported in two other round-robin studies for standard
solutions: 18 to 61% and 8 to 43% [33,34]. This indicates that
the determination of dioxin-like activity in sediment using the
DR CALUX bioassay is at least as consistent as the established
HRGCMS methods. In addition, the results show that the intra-
and interlaboratory repeatability is comparable. From the data
it can also been seen that the repeatability was lowest for phase
3, during which the participants were asked to extract, clean
up, and perform a DR CALUX bioassay on a suppliedsediment
sample. Since in the third phase, extra steps to the total pro-
cedure are introduced (extraction and cleanup), it is very likely
that these add to the variability of the total process. Further-
more, as none of the participants had prior experience using
the supplied extraction procedure, it can be anticipated that
with increasing experience using the supplied extraction pro-
tocol, the repeatability will also increase.
Reproducibility
As with the determination of the intralaboratory repeat-
ability, the intralaboratory reproducibility was determined by
analyzing a cleaned sediment extract and a 3-pM 2,3,7,8-
TCDD standard on 10 separate days and by multiple persons.
The reproducibility for the 3-pM 2,3,7,8-TCDD standard was
found to be 13.8%, whereas the reproducibility for the cleaned
sediment extract was shown to be 19.9%. Since the observed
reproducibilities are in the range of relative standard deviations
for two sediment extracts analyzed in 10-fold on the same day
(intralaboratory repeatability), the DR CALUX bioassay can
be evaluated as a stable and robust bioanalytical tool.
The interlaboratory results obtained from the analysis of
defined standard solutions, but also from the analysis of sed-
iment extracts prepared either by the coordinator of the study
or by the participants themselves, also provide a measure of
the variation between laboratories. The results show that the
interlaboratory reproducibility ranges from 6.5% for the de-
fined dioxin sample to 27.9% for the sediment sampleextracted
by the participants themselves. As was mentioned before, the
reproducibility for this last sample is relatively high and most
presumably due to the introduction of extra handlings (ex-
traction and cleanup) to the total procedure. In addition, the
fact that not all the participants had prior experience with the
extraction protocol to be used could have added to the increase
in variability of the process. Furthermore, the dilution factor
was not dictated. This also introduces a certain degree of var-
iation. For the reproducibility of the DR CALUX bioassay
itself and not caused by differences in operating extraction
conditions, the maximum variation between laboratories was
observed to be 18.0%. The results for the sediment extract
samples can also be used to estimate the method variability
for extracts, that is, based on samples of unknown composition.
Again, given the intra- as well as the interlaboratory variations
observed in this study, it appears justified to conclude that the
standard deviation of the means provides a reasonable estimate
of the method variability, based on the overall average con-
centrations (in 2,3,7,8-TCDD TEQs) for a single sediment ex-
tract sample, determined at two different dilutions. The largest
standard deviation of the means is therefore proposed as the
method error for analyzed samples, being 18.0% for sediment
extracts and 10.5% for analytical samples.
CONCLUSION
Several overall conclusions can be drawn based on the sta-
tistical evaluation of the data submitted by the participants of
the DR CALUX intra- and interlaboratory validation study.
First, differences in expertise between the laboratories are ap-
parent based on the results for the calibration curves (both for
the curves as provided by the coordinator and for the curves
that were prepared by the participants) and on the differences
in individual measurement variability. Second, the average re-
sults, over all participants, are very close to the ‘‘true’’ con-
centration, expressed in DR CALUX 2,3,7,8-TCDD TEQs for
the analytical samples. Furthermore, the interlaboratory vari-
ation for the different sample types can be regarded as esti-
mates for the method variability. The analytical method var-
iability is estimated to be ?10.5% for analytical samples and
?22.0% for sediment extracts. Finally, responses appear de-
pendent on the dilution of the final solution to be measured.
This is hypothesized to be due to differences in dose–effect
curves for different dioxin responsive element–active sub-
stances. For 2,3,7,8-TCDD, this effect is not observed.Overall,
based on bioassay characteristics presented here and harmo-
nized quality criteria published elsewhere [27], the DR CA-
LUX bioassay is regarded as an accurate and reliable tool for
intensive monitoring of coastal sediments.
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