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Chemosphere. Author manuscript; available in PMC 2015 January 28.

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Published in final edited form as:
Chemosphere. 2014 January ; 94: 27–35. doi:10.1016/j.chemosphere.2013.08.075.

Time-integrated Monitoring of Polychlorinated Dibenzo-pdioxins and Polychlorinated Dibenzofurans (PCDD/Fs) in Urban
and Industrial Wastewaters Using a Ceramic Toximeter and the
CALUX Bioassay
Amr Addecka,*, Kim Croesa, Kersten Van Langenhovea, Michael S. Denisonb, Ahmed
Elhamalawyc, Marc Elskensa, and Willy Baeyensa
aDepartment of Analytical and Environmental Chemistry, Vrije Universiteit Brussel (VUB),

Pleinlaan 2, 1050-Brussels, Belgium
bDepartment of Environmental Toxicology, University of California at Davis, Davis, CA 95616,

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cDepartment of Physics, Faculty of Science, Menofia University, Shebin Elkoum, Egypt

Abstract

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The ceramic toximeter as a passive sampler in combination with the CALUX bioassay wasutilized as a time-integrated pollution-assessment technique for dioxin-like PCDD/Fs in
wastewaters. Toximeters filled with XCARB and enclosed in stainless steel cages were submergedin wastewater of Belbeis drainage-canal and EMAK paper-mill, located in Egypt, for 28 days.
Two samplers were removed every week from each site. Extracts from toximeters, from bottomsediments, and from paper-mill sludge were analyzed by the CALUX. Results showed a regularincrease in the amounts of PCDD/Fs trapped by the toximeters as sampling time increased. Timeweighted average concentrations (TWA) of PCDD/Fs in the wastewaters of the drainage-canal andpaper-mill were 231 (214-281) and 26 (24.1-32.6) pg-BEQ L-1, respectively. Compared toliterature data, this means a high pollution level for the drainage-canal water, while the pollution
level in the paper-mill wastewater is similar to that reported for other paper mills. The PCDD/Fs
levels in the drainage-canal bottom-sediments and in the paper-mill sludge were 8.3 and 14.6 pgBEQ g-1 dry-weight, at the lower end of internationally reported values in similar compartments.
The sampling rate of the tested toximeters for dissolved PCDD/Fs was about 3.6 mL d-1.
Sediment/water partitioning coefficient log Kd of PCDD/Fs in the drainage-canal and the papermill was 1.42 and 2.70 respectively. The organic-carbon normalized partition coefficient log Koc
was 4.17 and 3.19 respectively, and is lower than the reported values for other sites.

© 2013 Elsevier Ltd. All rights reserved.
*

Corresponding author: Tel: +32 2629 3602; Fax: +32 2629 3274. aaddeck@vub.ac.be (A. Addeck).
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to ourcustomers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of
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Keywords

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Passive sampling; Ceramic toximeter; CALUX; PCDD/Fs; Wastewater monitoring; Partitioning
coefficient

1. Introduction

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A wide variety of synthetic chemicals are continuously released into the environment during
production, usage, and disposal of products. Physicochemical properties of these man-madechemicals are wide-ranging, and so are their effects. Amongst them, polychlorinateddibenzo-ρ-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), pose significant
threats to human health and the environment (Kassim and Barceló, 2009). They have beenfound in all compartments of the ecosystem, including water, although their aqueous
solubility is very low (Van den Berg et al., 2006). In addition to their environmental hazards,
PCDD/Fs accumulate in the fat tissue of animals and humans causing potent carcinogenic
effects, endocrine-disruption, reproductive, developmental and neurotoxic effects (Wikoff etal., 2012). Monitoring the presence and quantity of these compounds in the environment ona regular basis is therefore not a question of choice, but merely a necessary action for better
health protection (Seethapathy et al., 2008).

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Passive sampling techniques are gaining an increasing interest in pollution assessment inseveral environmental compartments. A passive sampler is able to acquire a sample without
any active media transport induced by pumping or purging techniques. Based on transport
process differences through the housing and the receiving phases, two types of passive
samplers can be classified: (1) equilibrium and (2) non-equilibrium based passive samplers(Kot-Wasik et al., 2007). Equilibrium samplers are characterized by a rapid achievement ofequilibrium state between contaminants in the water to be sampled and contaminants inside
the passive sampler. One consequence of achieving the equilibrium rapidly is that
contaminants are also capable of diffusing back into the surrounding water when the
aqueous concentrations of contaminants decline. Non-equilibrium samplers are those that do
not reach equilibrium with the surrounding water within the sampling period. Thesesamplers are characterized by a high capacity for collecting the contaminants of interest,
which in turn ensures that contaminants can be enriched continuously throughout the
sampling period (Kot-Wasik et al., 2007). The ceramic toximeter belongs to the latter ones
and is the subject of this research.
The ceramic toximeters are based on the free flow of analyte molecules, from the sampled
medium through a ceramic diffusion membrane, towards a suitable receiving medium underthe effect of a chemical-potential difference (Weiß et al., 2007). This flow of analyte(s)continues until equilibrium is established in the system or until the desired sampling period
is reached. The receiving phase should be selectively chosen depending on the analytes ofinterest. It must act as a perfect sink, which means that it must not release the trappedmolecules even if the concentration of the analytes around the sampler decreased to zero
(Weiß et al., 2007). The scientific validity and cost effectiveness of using this technique forvolatile organic compounds (VOCs) in water had been previously documented (Bopp et al.,2005; Verreydt et al., 2010). In a recent laboratory study, we validated the use of thisChemosphere. Author manuscript; available in PMC 2015 January 28.

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sampler for dioxin-contaminated water (Addeck et al., 2012). While the relatively low
sampling rate necessitates long sampling times when the concentrations are low, it does
enable in a simple way (only one analysis is necessary) the assessment of TWA
concentrations over long periods (Weiß et al., 2007).
We have used the CALUX (Chemically Activated LUciferase gene eXpression) bioassay for
determining the PCDD/Fs levels trapped in the toximeters as well as their levels in thesediments and sludge collected from the sampling sites. The CALUX technique makes useof a reporter gene expressed by recombinant mammalian cells. The genetically-modified
cells used contain a stably transfected AhR-responsive firefly luciferase reporter gene,which responds to dioxin-like chemicals by the induction of luciferase in a time-, dose-, andAhR-dependent manner (He et al., 2011). The CALUX signal (gene-induced light intensity)
is quantified through a BEQ-value; a Biochemical EQuivalent expressed relative to the most
toxic dioxin congener 2,3,7,8-TCDD. The advantage of CALUX is to assess directly the
PCDD/Fs level present in the sample in “Toxic Equivalent Quantity” without going throughcalculations based on analysis of complex chemical and toxic equivalency factors (Campell
et al., 2006).

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The ceramic toximeter in combination with the CALUX bioassay was previously validated
as an analytical tool for the determination of PCDD/Fs in solution (Addeck et al., 2012).
However, concentrations of dissolved PCDD/Fs in real aquatic systems are very low whichmakes their in situ determination a real challenge. Hence, the first goal of this research was
to apply the passive sampler in aquatic systems that could be contaminated by PCDD/Fs andto determine their concentrations: for that purpose a drainage-canal system and a paper milleffluent, both impacted by industrial and municipal activities (see sampling sites here below)
were selected. In addition, the amount of PCDD/Fs accumulated by the passive sampler
should increase linearly with the period of exposure. This has been verified too.

2. Materials and Methods
2.1. Sampling Sites

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2.1.1. Drainage-canal system—Sampling was carried out during October 2011 at aselected point in Belbeis drainage-canal (N: 30° 26′ 25.88″, E: 31° 34′ 23.16″), east side of
the Nile delta, (Egypt). The surrounding area consists of cultivated and inhabited land (Fig.
1). The canal is frequently used to convey raw industrial and municipal wastewater. The
entire system is heavily polluted, mainly by sewage. The drainage system and its mouth at
Lake El-Manzala are identified as “black spots” starting from 1992 (DRI, 2005) and are still
representing a big environmental challenge. During the sampling period, water temperature
ranged between 23 – 25 °C. The water-stream flows at 1.7 km/h at the sampling point, thebasin depth was about 2 m and its surface width is about 35 m. The color of the water was
black and its bad smell likely drives from its very high concentration of sulfides (23.5 mg
L-1) (EAA, 2011). Illegal burning of rice-straw was observed nearby the drainage-canalshores and in surrounding farms. This act is a major source of air and water pollution in thearea (MSEA, 2011).

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2.1.2. Paper-mill effluents—Sampling in effluents of the EMAK paper-mill (N:
29°41′50.12″, E: 32°18′13.30″), Ain El-Soukhna, Egypt, was carried out during October
2011. The mill produces 60,000 tons/year of double-coated duplex paper board. About 95%of the raw materials in production consists of recycled paper of all kinds, while theremaining percentage is new pulp from other suppliers. The toximeters were tested at two
points inside the factory: (1) Main collecting pool: contains water mixed with fine fibers
produced from the milling process. (2) Biologically-treated wastewater pathway. Water
temperature ranged between 24-26 °C during the sampling period.
2.2. Chemicals and materials

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The ceramic cylinders were obtained from ATECH Innovations (Germany). Acetone (PestiS grade, minimum 99.9%), hexane (for dioxins and PCBs, minimum 96%) and toluene (fordioxins and PCBs, minimum 99.8%) were from Biosolve (The Netherlands). The XCARB(activated carbon/celite 1% w/w) was from Xenobiotic Detection Systems (USA). Neutral
alumina (activated, 150 mesh), silver nitrate (5 wt % on silica gel 60), ethyl acetate pestanaland silica gel 60 were from Sigma–Aldrich (Germany). Sulfuric acid (95–97%, ACS
reagent), DMSO and glass-wool were obtained from Merck (Germany). Anhydrous sodium
sulfate was from Boom (The Netherlands). Glass-fiber filters for ASE were from Whatman(UK). Standard solution of 2,3,7,8 TCDD (50 ng ml-1) was from Campro Scientific (TheNetherlands). The mouse hepatoma H1L7.5c1 cell line was provided by Michael Denison(University of California, USA).
2.3. Conditioning of materials
The ceramic cylinders were sintered at 750 °C for 24 hours to eliminate possible organic
traces. Glassware was baked for 4 hours at 450 °C. XCARB and glass-wool wereconditioned by fluxing in toluene in an ultrasonic bath for 1 hour. Toluene was decanted andthe materials were rinsed twice with fresh amounts of toluene before drying. Silica gel was
baked at 200 °C for 48 h. PTFE caps and stainless steel cages were rinsed with acetone anddeionized water.
2.4. Sampling kits and exposure scenario

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A total number of 18 toximeters were used to sample PCDD/Fs for a total period of 28 days.
Weekly, 2 toximeters (for duplicate samples) were removed from each sampling site to test
the linearity between the exposure period and the mass of trapped PCDD/Fs. Each toximeterconsists of a ceramic cylinder of alumina (α-Al2O3) lined with a 5 μm coating of titania(TiO2). The cylinders were 8 cm length (only 7 cm are exposed for diffusion), 0.2 cm wall
thickness, 1 cm external diameter, and 30% porosity. The mean pore diameter of thealumina membrane was 0.4 μm and that of the titania coating was 0.05 μm. The titania
coating serves as a diffusion barrier limiting the movement of analytes to molecular
diffusion only (Fig. 2) (Addeck et al., 2012). The pore diameter was small enough to preventcrossing or growth of microorganisms, as the most dominant bacteria in water systems are
0.5–5.0 μm (Sigee, 2005). A mass of 1.15 g of pre-conditioned XCARB was packed in eachcylinder and small plugs of pre-cleaned glass-wool were inserted in both ends. PTFE caps
tightly closed both ends of the cylinders. Toximeters were kept in light-shielded glass bottles

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filled with Milli-Q deionized water till the time of deployment. The bottles were vacuumed
to ensure full saturation of toximeters with water. Cages of stainless-steel grids were used to
enclose and protect the fragile samplers (4 samplers/cage) (Fig. 2). The cages' grid reduces
accumulation of the suspended particles on samplers' surface. Cages were loaded withtoximeters just before application, and suspended in the water column in both sites. Bottomsediments and fibrous sludge were collected from the drainage-canal and the paper-mill at
the end of sampling period. Sediments, sludge and toximeters were light-shielded andpreserved at -20 °C till the time of extraction. Two full toximeters were kept in deionizedwater as blanks.
2.5. Extraction of toximeters, sediments, and sludge

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The toximeters were rinsed with deionized water and left to dry. Caps were removed and the
XCARB was ejected into glass vials. Toluene was used as extraction solvent as it gives thehighest recovery rates for PCDD/Fs from XCARB (XDS, 2008). First, 15 mL of toluene wasadded to the vials. Then, the samples were ultrasonicated, centrifuged to settle down thesuspended particles, and the liquid phase was pipetted into new glass vials through precleaned glass-wool filters. This step was repeated 3 more times for all samples using 9, 9,
and 6 mL of fresh toluene (Addeck et al., 2012). The drainage-canal sediments and papermill sludge were freeze-dried in a lyophilizer. They were homogenized with a pestle andmortar. Liquid nitrogen was added during paper-mill sludge processing to assist the breakupof the fibrous blocks. The homogenized powders were then sieved (35 mesh) to remove thelarger grains such as pebbles and blocks. Five grams of both sediments and sludge wereextracted using pressurized liquid extraction (ASE 200, Dionex Sunnyvale, CA, USA)equipped with 33 mL stainless steel extraction cells. A mixture of hexane/acetone (1/1, v/v)
was used as an extraction solvent because it gave reliable results compared to other solventsfor fish, meat, atmospheric deposition and biological samples (Croes et al., 2011). The
extraction conditions were 125 °C, 2 cycle extraction, 6 min heating time, 10 min static
period, 1500 PSI pressure, and 60% flushing volume. Afterwards, the extracts were
evaporated and the residues were dissolved in 10 ml of hexane.
2.6. Clean-up of extracts

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All extracts from toximeters, sediments, and sludge were pretreated by adding 4 mL sulfuric
acid. The clean-up took place through a multi-layer silica gel column coupled in series witha carbon column to separate PCDD/Fs from dioxin-like PCBs and other compounds thatmight interfere during the CALUX bioassay analysis. The silica gel column (25 mL) was
filled from bottom to top with a glass-wool plug, 1.9 g sodium sulfate, 2.0 g deactivatedneutral alumina, 3.0 g of a 33% (w/w) sulfuric acid on silica gel mixture, 1.0 g silver nitrate(5 wt% on silica gel 60), 2.8 g of a 33% (w/w) sulfuric acid on silica gel mixture, and 1.9 g
sodium sulfate. The carbon column (10 mL) was filled from bottom to top with a glass-wool
plug, 0.7 g sodium sulfate, 0.34 g XCARB, and 0.7 g sodium sulfate (Figure 3). Before
loading the samples, the acid silica columns were rinsed with 60 mL hexane, and the carbon
columns were rinsed with 5 mL acetone, 20 mL toluene, and 10 mL hexane (Croes et al.,2011). After adding the samples, the columns were rinsed with 45 mL hexane, and then thesilica columns were removed. The carbon columns were first rinsed with 8 mL hexane/
acetone (9/1) and the coplanar PCBs were eluted with 15 mL hexane/ethyl acetate/toluene
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NIH-PA Author Manuscript(80/10/10). Finally, the PCDD/Fs were eluted with 20 mL toluene, centrifuged undervacuum (40 °C) till dryness, and dissolved in 4 mL hexane. A dilution series was made foreach sample prior to the CALUX analysis. A schematic overview of the sampling strategy,
extraction, clean up, fractionation and final measurements is given in Figure 3.
2.7. The CALUX bioassay

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The CALUX bioassay was performed using the sensitive mouse hepatoma H1L7.5c1 cellline. The applied protocol was previously described by Van Langenhove et al., (2011) and isbased on U.S. EPA method 4435 (XDS, 2008). Briefly, the cells were grown in cell culture
plates containing 15 mL RPMI 1640 supplemented with 8% FCS and 1% penicillin/
streptomycin (Gibco, UK). After trypsinizing, the cells were counted and diluted to aconcentration of 55-65×104 cells mL-1 (Croes et al., 2011). Each well on the 96-well plate
was seeded with 200 μL cell suspension in RPMI. After 24 h incubation at 37 °C with 5%
CO2 and 80% humidity, 188 μL of a standard solution or sample extract in RPMI with 1%
DMSO was added to each well. After 20-24 h incubation period, the medium was removed
and the wells were rinsed with 75 μL PBS buffer (Gibco, UK). Then the plate was visuallyinspected under the microscope for any significant defects in cell morphology. 50 μL lysis
reagent (Promega, USA) was added and the plate was shaken for 5 min. After a 10 min
incubation period in the luminometer (Glomax, Promega, USA), 50 μL luciferine reagent(Promega, USA) was injected into all wells (integration time 3 s, lag time 5.6 s) The
measured luminescence was expressed in relative light units (RLU) which were later
converted into bioanalytical equivalency value (CALUX-BEQ) by comparing the responseof a given sample to a dose-response curve obtained from 2,3,7,8-TCDD standards (Croes etal., 2011). Results from CALUX are expressed in pg-BEQ L-1 while the GC-HRMS
literature data (to compare with) are presented as toxic equivalency values (TEQs) and
expressed in pg-TEQ L-1.
2.8. Total organic carbon (TOC) content in sediments and sludge

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The total organic carbon (TOC) content in bottom-sediments and fibrous sludge samples
was measured so that the organic-carbon normalized partition coefficient for PCDD/Fs inboth aquatic environments could be calculated. TOC measurements were carried out with a
CHN elemental analyzer (Flash 1112 EA Elemental Analyser, Thermo Finnigan, Italy). The
organic matter in the dry homogenized samples was quantitatively converted to CO2 N2,
SO2, and H2O by high temperature oxidative combustion at >900°C in an O2 atmosphere,
after removal of the inorganic carbon in the sample by acidification with 5% HCl. The EA
analyzer is connected to a gas chromatograph that separates the combustion gases. TOC
quantification was performed by comparing the CO2 signal with that of an aniline standard.
2.9 QA/QC
On all 96-well plates, a 10-point TCDD calibration curve, three DMSO blanks and three
quality control (QC) solutions (a TCDD standard solution with a concentration of 0.125 ng
mL-1) were added in duplicate. The minimum detectable value was calculated as theconcentration level at the inflection point of the TCDD curve where the slope is significantly
different from zero (i.e. different from the lower plateau of the sigmoid curve), using a t-test(p=0.01) (Croes, 2012).
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Also two blank toximeters (saturated with ultrapure water) were analyzed as a quality
control for the whole analytical procedure. The blank extracts were spiked with the QC
solution before measurement with the mouse cell line to take into account possible
antagonistic effects. Recovery of the spiked blanks should range between 80 and 120%.

3. Results and Discussion
3.1. QA/QC results
All TCDD calibration curves were fitted with a 4-parameter Hill Equation, using a Weighted
Least Squares (WLSs) regression technique (Elskens et al., 2011). The mean EC50 value(n=6) yielded 565 pg per well (SE=46 pg per well). The minimum detectable value was
0.046 pg BEQ per well. All quality control solutions and the toximeter blanks yielded
recoveries between 80-120%, while the DMSO blanks were at background level (lower
plateau of the calibration curve).
3.2. Quantification of extracted PCDD/Fs

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The amounts of accumulated PCDD/Fs were found to increase in toximeters with increasing
sampling time (Table 1). This increase was linear except for the results obtained during the
4th week with the drainage-canal samplers (Fig. 4 and Eq. 1 and 2).

(Eq. 1)(Eq. 2)

With y=total mass accumulated on the taximeter (pg BEQ/taximeter) and x= exposure time(days)

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The lower amount of PCDD/Fs observed in the drainage-canal water during the last week
compared to the previous weeks can be due to several causes. The first possibility is that the
XCARB (the receiving medium), has become saturated. However, the adsorption capacity of
the XCARB towards dioxins is at least 400 times higher than the highest extracted amountper toximeter (capacity is > 0.1 μg-TEQ PCDD/Fs g-1) (Chi et al., 2006). Even if otherorganic pollutants may also be adsorbed to the XCARB, its adsorption capacity is very highand seems to be sufficient. The second possibility is a sudden change in the levels of
dissolved chemicals in the drainage-canal water. This could be due to an irregular andmomentary discharge of some specific waste from -for example- a gas station close to thesampling area. Wastes from the latter normally contain a variety of petrogenic hydrocarbons
that may disturb the water/sediment equilibrium by affecting the distribution ratio ofhydrophobic chemicals in the system. Monitoring records show values between 0.2 and 1.7
μg L-1 of petrogenic hydrocarbons along the drainage system in May and August 2011
(EAA, 2011). The third possibility is a reduction in the adsorption rate of PCDD/Fs onto the
XCARB, due to competitive binding in the drainage-canal water by -for example- activecarbon resulting from rice-straw burning alongside the drainage-canal shores which iscommon during this period of the year.

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Overall, the results obtained after one, two and three weeks showed a linear increase in theamount adsorbed by the toximeters and allowed a correct estimate of the PCDD/Fs levels inthe drainage-canal water over that period. During the fourth week the adsorption rate in thetoximeters decreased for an unknown reason as described above. This observation might bea useful indicator on some momentary events that could be over- or underestimated by theconventional snapshot sampling technique. The effect of environmental conditions, like
temperature, was considered insignificant as it had changed only slightly during thesampling period. A temperature increase of 1 °C corresponds to an increase in the diffusion
coefficients (Dw) of about 3-4% (Weiß et al., 2007).
3.3. Calculation of the time-weighted average concentration (TWA) of PCDD/Fs (pg-BEQ
L-1)

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The CALUX analysis yielded the mass M (pg) of PCDD/Fs that passed through theavailable surface area A (21.98 cm2) of the ceramic membrane over the sampling period t (7,14, 21 and 28 days) and trapped by XCARB (Table 1). Assuming that the transport of the
PCDD/Fs to the inner part of the ceramic membrane through the diffusion path Δx (0.2 cm)
was based solely on molecular diffusion, the time-weighted average concentration of thesechemicals in the sampled medium Cw (pg-BEQ L-1) can be derived from Eq. (3), in thiscase, calibration and frequent snapshots are not necessary (Weiß et al., 2007):(Eq. 3)

Where De (cm2 s-1), the effective diffusion coefficient, refers to the alteration in the
diffusion rate of analytes (PCDD/Fs) in the porous ceramic membrane compared to that ofwater. It can be calculated from Archie's law Eq. (4) (Weiß et al., 2007):(Eq. 4)

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Where Dw (cm2 s-1) is the diffusion coefficient of PCDD/Fs in water, ε is the porosity of theceramic membrane (30%), and m is Archie's law exponent, which ranges between 1.5 to 2.5
in laboratory calibration experiments (Weiß et al., 2007). A value of 2.0 was previously
determined and used in field applications with ceramic-based passive samplers (Bopp et al.,2005; Martin et al., 2003). Since there are no direct measurements for aqueous diffusivity of
PCDD/Fs, we have used an empirical formula Eq. (5) (U.S. EPA, 1987) to calculate thediffusion coefficient in water Dw for all PCDD/F congeners classified by the World HealthOrganization (WHO) for their dioxin-like toxicity.

(Eq. 5)

Where Mwt (g/mol) is the analyte's molecular weight. Results are shown in annex. The
average value for De is 3.79 ×10-7 cm2 s-1 with a minimum of 3.32 ×10-7 cm2 s-1 and amaximum of 4.36 ×10-7 cm2 s-1.

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By applying all parameters to Eq. (3) and using the mean De value, the mean concentrationsof PCDD/Fs in water during the sampling period (TWA) of Belbeis drainage-canal and
EMAK paper-mill were 231 and 26 pg-BEQ L-1, respectively. Using the minimum andmaximum De values, we obtain for the drainage-canal concentrations of 214 and 281 pgBEQ L-1 and for the paper-mill 24.1 and 32.6 pg-BEQ L-1. Since on the international levelno concentration limits for PCDD/Fs in aquatic systems were established, we compared our
results with those from other studies.
In Newark Bay (New Jersey, USA), dissolved PCDD/Fs concentrations ≤ 20 pg-TEQ L-1
were recently observed using polyethylene (PE) passive samplers (Friedman et al., 2012).
An earlier study on PCDD/Fs in UK rivers revealed a concentration of 80 pg-TEQ L-1 (Roseet al., 1994). Water samples taken after the 2002 flood in the Elbe River showed PCDD/F
levels from 0.1 to 14.7 pg-TEQ L-1 in the Czech Republic (Holoubek et al., 2003) and from
4.4 to 15.5 pg-TEQ L-1 (Wittenberg) and 37.0 to 85.9 pg-TEQ L-1 (Magdeburg) in Germany(Theile et al., 2003). Comparing our PCDD/Fs levels in the Belbeis drainage-canal withthose from literature, we must conclude that the canal is heavily polluted with PCDD/Fs.

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Regarding PCDD/Fs levels in the wastewater from paper-mills, in Sweden, concentrations
between 0.11 and 133 pg-TEQ L-1 with a mean value of 19.1 pg-TEQ L-1 were reported(Buckley-Golder et al., 1999). In China, a study on PCDD/Fs levels in wastewater from alarge pulp-mill, showed an average concentration of 315.6 pg-TEQ L-1 (Zheng et al., 2001).
The PCDD/Fs levels in the industrial water of the EMAK paper-mill are in accordance withthose literature values. The toximeters from the biologically-treated wastewater of EMAK
paper-mill did not show a decrease in PCDD/Fs content (data not shown). This was expected
because the applied biological treatment process is not basically meant to remove this kindof chemicals.
3.4. Sediments and sludge

NIH-PA Author ManuscriptCALUX analyses of the bottom-sediments and fibrous sludge yielded PCDD/Fs contents of
8.3and 14.6 pg-BEQ g-1 dry-weight, respectively. For comparison, the levels of PCDD/Fs in
the sediments from 3 industrial areas in Cairo, Egypt, were between 1.8 and 38 pg-TEQ g-1
d.w. (El-Kady et al., 2007). In 2001, PCDD/Fs levels of 1.64 and 1.83 pg-TEQ g-1 d.w.,
were reported for two regions with industrial sites (Zlín and Beroun) in Czech Republic(E.C., 2004). In the Netherlands, sediments of rivers, lakes, and canals showed PCDD/Fs
levels between 1 and 10 pg-TEQ g-1 d.w. (Buckley-Golder et al., 1999). In Belgium,
CALUX measurements of PCDD/Fs in sediments of the Yser and the Scheldt Rivers showedvalues between 10 and 42 pg-BEQ g-1 d.w. (Sanctorum et al., 2007). In general, the typical
PCDD/Fs contamination levels of the sediments in the EU member states range from < 1 up
to 200 pg-TEQ g-1 d.w. (Buckley-Golder et al., 1999). When comparing these values withthe measured PCDD/Fs levels in the Belbeis drainage-canal sediments and EMAK papermill sludge, it appears that they are within the typical European levels.
The relatively low PCDD/Fs content in the drainage-canal bottom-sediments compared totheir dissolved levels in water column, and to other much less contaminated sites might be
attributed to the high content of petrogenic hydrocarbons in the drainage-canal water. The
latter compete with the sediments and especially its organic fraction for the PCDD/Fs
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present in the system and thus affect their sediment/water partition ratio. The hydrocarbon
levels varied from 0.53 to 1.74 μg L-1 in May 2011 and 0.20 to 1.24 μg L-1 in August 2011,
along the drain-mouth, Lake El-Manzala (EAA, 2011).
3.5. Distribution coefficients in the two sampling environments
The sediments/water partitioning (or distribution) coefficient of PCDD/Fs in both samplingsites (a site-specific parameter) was calculated according to Eq. (6) (Suarez et al., 2006):(Eq. 6)

Where: Koc: organic-carbon normalized partition coefficient (between the solid organic
matter phase and the dissolved phase) for PCDD/Fs congeners (annex), Kd: distribution
coefficient between the solid phase and the dissolved phase for PCDD/Fs, foc: measured
mass fraction of organic carbon (TOC) in the solid-phase, Cs: measured concentration of
PCDD/Fs in dry solid-phase sediments or sludge (pg-BEQ g-1), and Cw: measured
concentration of PCDD/Fs in the dissolved phase (pg-BEQ L-1).

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Solving Eq. (6), log Kd amounts to 1.42 and 2.70 for the drainage-canal and paper-mill
wastewater, respectively, while using the measured TOC content in bottom-sediments(canal) of 0.18 % and in sludge (paper-mill) of 32.5 %, log Koc amounts to 4.17 and 3.19,
respectively. The value of log Koc in the drainage-canal is much lower than the values
reported for PCDD/Fs congeners in other aqueous environments (annex). This might be
attributed to a disturbed sediments/water partitioning due to the reported presence of
petrogenic hydrocarbons in the canal water (EAA, 2011).
3.6. Sampling rates of the ceramic toximeter for freely-dissolved PCDD/Fs
The sampling rate describes the volume of water that is extracted with regard to thechemicals of concern within a certain period of time. In the case of a ceramic membrane,
sampling rates are defined according to Eq. (7) (Weiß et al., 2007):(Eq. 7)

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Applying the calculated mean value of De for dioxin-like PCDD/Fs, the sampling rate of theceramic toximeters for PCDD/Fs is about 3.6 mL d-1. The low sampling rates of ceramic
toximeters emphasize their suitability for long-term, time-integrated monitoring. The
downside of the low sampling rate is that extended time periods are required to reach thedetection limits of the analytical technique, especially in low contaminated environments.

4. Conclusion
In this study, we have applied the ceramic toximeter combined with the CALUX bioassay
technique to measure time-weighted average concentration (TWA) (pg-BEQ L-1) of
PCDD/Fs in urban and industrial wastewaters. The idea is based on accumulating chemicals
of interest (PCDD/Fs) using a suitable receiving phase (XCARB) that maintains a
concentration gradient across the diffusion membrane (ceramic). The amounts of

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accumulated PCDD/Fs extracted from toximeters were measured by the CALUX semiquantitative technique.

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Results from the Belbeis drainage-canal and the EMAK paper-mill, Egypt, showed a linear
intake of dioxin-like PCDD/Fs with time, with an unexplained decrease during the fourthweek for the drainage-canal samplers. The average (minimum-maximum) PCDD/Fs levels
in both sampling sites during the sampling period (28 days) were 231 (214-281) and 26(24.1-32.6) pg-BEQ L-1, respectively. The levels of PCDD/Fs observed in the drainagecanal bottom-sediments and the paper-mill fibrous sludge were 8.3 and 14.6 pg-BEQ g-1
dry-weight, respectively. Comparison with data available for other sites was made. It
appears that the drainage-canal water is heavily polluted with PCDD/Fs while the paper-mill
wastewater is within the reported values for this kind of industry. The low levels of
PCDD/Fs in the canal bottom sediments might be attributed to the reported high content of
petrogenic hydrocarbons in the canal water, which compete with the sediments for dioxins.
Sediment/water partitioning coefficient log Kd of PCDD/Fs in the canal and the paper-mill
was 1.42 and 2.70 respectively. The log Koc was 4.17 and 3.19, respectively, and lower thanpreviously reported values for other sites.

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The sampling rate of the ceramic toximeters for PCDD/Fs was found to be 3.6 mL d-1,
which is low compared to other passive samplers. The low sampling rate might also be
considered an advantage as it enables long-term and time-integrated pollution assessment forsuch POPs in aqueous environments, making it a relatively inexpensive method to routinely
study the levels, behavior, and fate of these pollutants in aqueous systems.

Acknowledgments
This work was supported by Erasmus-Mundus program (EMECW) through a grant attributed to A. Addeck. The
H1L7.5c1 cell line was developed by M. Denison with funding from a Superfund Research Program grant(ES04699) from the National Institute of Environmental Health Sciences. The help of Natacha Brion in TOC
measurement is appreciated. We would like also to thank Mohamed Ataalla, Gamal Atta and the staff of EMAK
paper company for their valuable cooperation, and Jean-Pierre Clement for manufacturing of the samplers'
housings.

Annex
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Calculated diffusion coefficients in water (Dw) and effective diffusion coefficient in ceramic
membrane (De) for dioxin-like PCDD/Fs. Molecular weight and log Koc data from (Mackay
et al., 2006).
Mwt (g/mol)Dw (cm2 s-1)

De (cm2 s-1)

log Koc

321.97

4.68×10-6

4.21×10-7

6.30

1,2,3,7,8-PnCDD

356.42

4.38×10-6

3.94×10-7

8.72

1,2,3,4,7,8-HxCDD

390.86

4.12×10-6

3.70×10-7

6.02

1,2,3,6,7,8-HxCDD

390.86

4.12×10-6

3.70×10-7

9.42

1,2,3,7,8,9-HxCDD

390.86

4.12×10-6

3.70×10-7

9.45

1,2,3,4,6,7,8-HpCDD

425.31

3.89×10-6

3.50×10-7

6.69

OCDD

459.75

3.69×10-6

3.32×10-7

10.96

Congeners
Dibenzo-p-dioxins
2,3,7,8-TCDD

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Page 12Mwt (g/mol)Dw (cm2 s-1)

De (cm2 s-1)

log Koc

305.984.85×10-6

4.36×10-7

5.18

1,2,3,7,8-PnCDF

340.42

4.51×10-6

4.06×10-7

8.03

2,3,4,7,8-PnCDF

340.42

4.51×10-6

4.06×10-7

5.59

1,2,3,4,7,8-HxCDF

374.86

4.23×10-6

3.81×10-7

7.40

1,2,3,6,7,8-HxCDF

374.86

4.23×10-6

3.81×10-7

7.40

1,2,3,7,8,9-HxCDF

374.86

4.23×10-6

3.81×10-7

9.08

2,3,4,6,7,8-HxCDF

374.86

4.23×10-6

3.81×10-7

6.80

1,2,3,4,6,7,8-HpCDF

409.31

3.99×10-6

3.59×10-7

6.00

1,2,3,4,7,8,9-HpCDF

409.31

3.99×10-6

3.59×10-7

5.00

443.75

3.78×10-6

3.40×10-7

6.00

Mean Dw, De (cm2 s-1) and log Koc

4.21×10-6

3.79×10-7

7.29

Congeners

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Dibenzofurans
2,3,7,8-TCDF

OCDF

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Ceramic toximeters were used as passive samplers for PCDD/Fs in wastewater.
Activated carbon/celite 1% (w/w) was chosen as a receiving phase to trap PCDD/Fs.
Urban and industrial wastewaters in two sites located in Egypt were tested.
CALUX bioassay was utilized as an analytical technique for extracted PCDD/Fs.
Results were compared to literature data from other sites worldwide.

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Fig. 1.

Sampling sites: (1) Belbeis drainage-canal and (2) EMAK paper-mill.

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Fig. 2.

A linear section of the ceramic toximeter and two stainless steel cages immersed in thedrainage-canal water for a duration of 1 week.

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Fig. 3.

Sample processing diagram for CALUX analysis.

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Fig. 4.

Increase in accumulated PCDD/Fs with sampling duration (drainage-canal samplers). Eachdata point is the mean value of duplicate samples.

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Table 1

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Mass (M) of extracted PCDD/Fs from toximeters and CALUX-BEQs for the sediments and sludge samples.
Two toximeters were analyzed at each time period (7 days) for both the Belbeis drainage-canal and EMAK
paper mill, except for the first time-period at the paper-mill (only one sample could be analyzed):
Drainage toximeters

Paper-mill toximeters

Time (days)

M Total PCDD/Fs (pgBEQ/toximeter) (SE)Total PCDD/Fs pr week
(pg-BEQ/toximeter) (SE)M Total PCDD/Fs (pgBEQ/toximeter) (SE)PCDD/Fs per week (pgBEQ/toximeter) (SE)7

6.28 (2.21)

6.28 (2.21)

0.83

0.83

14

13.6 (0.62)

7.27 (0.31)

1.37 (0.001)

0.54 (0.001)

21

19.5 (1.93)

5.91 (0.64)

1.96 (0.04)

0.59 (0.01)

28

21.0 (1.11)

1.55 (0.28)

2.51 (0.05)

0.55 (0.01)

Sediments/sludge

Drainage-canal bottom sediments 8.3 (SE=1.2) pg-BEQ/g
dry weight

Paper-mill fibrous sludge 14.6 (SE=2.1) pg-BEQ/g dryweight

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