Cod: 2.1037
ENDOCRINE ACTIVITY IN WASTE AND RIVER WATERS FROM THE BRUSSELS REGION,BELGIUM USING THE BG1LUC4E2 CALUX BIOASSAY.
K. Van Langenhove1, T. Reyns2, T. Vandermarken1, P. Servais3, M.S. Denison4, J. Van Loco2, M. Elskens1
1
Department of Analytical, Environmental and Geo-Chemistry (AMGC), Vrije Universiteit Brussel, Pleinlaan 2,1050 Elsene, Belgium
2
Wetenschappelijk Instituut voor Volksgezondheid, Rue Juliette Wytsmanstraat 14, 1050 Brussels, Belgium
3
Université Libre de Bruxelles, Ecologie des Systèmes Aquatiques, Av. Franklin Roosevelt 50, 1050 Brussels,Belgium
4Department of Environmental Toxicology, Meyer Hall, University of California, Davis, CA 95616, USA

Introduction
Today, there is international concern regarding the effects of natural and synthetic chemicals on thehealth of humans and wildlife since these emerging pollutants are able to interfere and act upon thehormonal system. These so-called endocrine-disrupting chemicals (EDCs) or endocrine active chemicals(EACs) are of particular concern to aquatic ecosystems, because these compounds are present in almost
all wastewater and treated wastewater effluents and in rivers receiving these effluents, ground watersupplies, sea water, sediment and biota, and could be of major concern for urban river systems such as
the Zenne River in Brussels, Belgium [1,2].
In this respect, the European Water Framework Directive (WFD) [3], whose main objective was to obtaina good ecological and chemical status for all European water bodies by 2015, established a priority list of
33 new and 8 previously regulated chemical pollutants presenting a significant risk to or via the aquaticenvironment. For the priority substances, environmental quality standards were set in 2008, and they
have to be monitored by all EU-Member States [3]. Several of these substances are recognized endocrine
disrupting chemicals such as the penta-bromodiphenylether (PBDE), octylphenol (OP), nonylphenol(NP) and the di(2-ethylhexyl)phthalate (DEHP), and are listed as hazardous priority substances [4]. In
addition, several (natural) hormones have been included in the WFD Watch List [3], among them 17ßestradiol (E2) and ethinylestradiol (EE2).
Rather than looking at individual concentrations of these compounds using hyphenated chromatography
techniques coupled to mass detection, biologically relevant and integrative approaches exist to perform
a first screening basis for endocrine activity [5,6]. For this reason, an in vitro trans-activational reporter
gene assay, BG1Luc4E2 cell line [7,8], was employed to carry out endocrine activity determination inboth Zenne River water, WWTP (waste water treatment plant) influents and effluents and also hospital
effluent from the Brussels Region.
The Zenne River itself is a small-sized river that is the receiving environment of treated wastewater from
2 major WWTPs in the Brussels area, Brussels North & South station, which are treating wastewaterof more than 1 million inhabitants. However, these WWTPs were not designed to deal with all thecontaminating compounds, and are not able to protect the Zenne, its ecological status and its inhabitants
against occasional contamination (such as point discharges, rain events…). This is especially true
for the WWTP South, which does not have tertiary and/or advanced treatment to specifically reduce
micropollutant load entering the Zenne ecosystem. The same applies for the WWTP North where tertiary
treatment is available, but only for the removal of nitrogen and phosphorus as micronutrients.
Specifically with regards to EACs, large inflows are provided by hospitals which release vast quantities
of wastewater containing EACs such as hormones, drugs and antibiotics and other pharmaceuticals andpersonal care products (PCPPs) [9] along with common municipal waste [10]. As a result, the Zenne
River, downstream of Brussels, is composed by more than 50% of WWTP effluents, and since there isno active treatment for removal of these compounds from the Brussels WWTP South water, nor from theWWTP North, it is of crucial importance to have an insight about the occurrence and endocrine activity
associated with these substances and following their path from hospital to Zenne River water.

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Materials and methods
A 12 month sampling program (January 2015 to February 2016) was carried out at 4 locations in theZenne River (Z3, Z5, Z9, Z11 according to literature [11]) taking 1L grab samples at each location. Inaddition, 24h composite samples were collected the day after at the WWTP North and South for in-/effluent as well as a grab sample of the raw hospital effluent (UZ Jette). The most upstream sampling site
is Z3, followed by WWTP South, Z5, WWTP North, Z9, and Z11 as most downstream [11]. Hospitalwastewater is diverted to WWTP North through the sewer system. In-situ analyses of pH, temp. (°C), O2content (mg O2/L), and conductivity (µS/cm) were recorded and logged. Samples (150-800mL) werefiltered within 24hrs using a 0.7µm glass fiber filter (GF/F Sartorius) and the filtrate was passed overan Oasis HLB (6cc 200mg glass) SPE cartridge to pre-concentrate estrogen target compounds according
to vendor protocol [12]. Samples were solvent exchanged to n-hexane for storage. Filters were dried toperform suspended particulate matter analysis [11].
CALUX (Chemically Activated Luciferase gene eXpression) analyses were carried out using theBG1 ERE TA cell assay (BG1Luc4E2 cell line) as described elsewhere [6–8]. Briefly, cells weremaintained in alpha minimal essential medium (α-MEM) supplemented with 10% (v/v) foetal bovine
serum (FBS). Media was exchanged to DMEM supplemented with L-Glutamine, Sodium Pyruvate,
Penicillin-Streptomycin and charcoal stripped FBS (i.e. estrogen free media) 48 hours prior to seedingin 96-well plates (200µL, density of 40,000 cells/well). Incubator settings were set at 37°C, 85% relative
humidity and 5% CO2. After an incubation time of 24hrs and cells reaching a monolayer, sample extract
dilutions and E2 treatment solutions (both 1% DMSO as final concentration) were dosed in triplicate(188µL). Cells were again incubated over a 24-hour period after which lysis and measurement wereperformed using Luciferase assay substrate and a Glomax 96-well plate reader (both from Promega,The Netherlands). Data analysis was performed in Excel® where statistical analysis and BEQ50/EC50
quantification [13] involved fitting the 4-parameter Hill equation. BEQ (as BEQ50) values are expressedin ng EEQ (Estrogen Equivalence)/L or otherwise as ng E2-eq./L (whereby E2 is the reference ligand
for this specific assay).
Results and discussion
Initial experiments conducted were aimed at optimizing and validating the method for estrogen analysis
using the in vitro reporter gene assay. Solvent blanks, matrix blanks (water), and recovery tests arerepresented in figure 1. These tests demonstrated that selecting appropriate solvent lots and water blanks
may be crucial in order to attain low background levels void of endocrine agonists. From figure 1 (leftgraph) it became apparent that the methanol contributed up to 100fg E2-eq./mL used. For comparison
reasons, the EC50 of E2 is approx. 526fg E2/well (n:180), indicating the need for a different solvent
lot that would meet the criterion of low agonist activity. A similar case is seen with blank water wheresome commercially available water displays high agonist activity (figure 1, left graph), even for MilliQ
treated water. However, carefully designing experiments (total volume of solvent or water) during blankand recovery tests with the lowest contributing solvents allows their combined agonist activity to be<LODDMSO of CALUX (approx. 64fg E2-eq./well (n:180)).
Recovery testing of E2 (figure 1 on the right) dissolved in water and extracted using Oasis HLB
SPE cartridges shows excellent results (individual recoveries ranging from 86-109%) and this over anenvironmentally relevant level (2-16ng E2/L).
After method optimization, a 12 month sampling program was performed with 9 samples analyzed
every month (4 Zenne River samples, 2 WWTP influent and effluent samples (4 in total), and 1 hospitalgrab sample). During a first series of tests, SPM results were analyzed (figure 2). As expected, Zenne
River values show the lowest amounts of suspended particulate matter (median of 21.4mg/L, averageof 34.6mg/L, and a Min-Max ranging from 2.31 to 314mg/L), followed by WWTP effluent (medianof 9.93mg/L, average of 36.46 mg/L, and ranging from 3.44 to 574mg/L). Both hospital and WWTP
influent yield high SPM content (respectively 252 and 329mg/L on average). The average reduction ofSPM content amounts to 93% in WWTP South and up to 96% in the North plant. However, an interesting
phenomenon is encountered during the 4th sampling campaign (figure 2), whereby extremely high levelsof SPM were measured in the WWTP influent and effluent and location Z5 (downstream of the WWTPSouth). The former results are indicative of a combined sewer overflow (CSO) event [11] whereby thesewer discharge exceeds the treatment capacity of the WWTP South and untreated wastewater is diverted
into the Zenne River.

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Such a CSO event is also noticeable when comparing BEQ values for endocrine activity among thedifferent sites (spatial) and sampling dates (temporal). Applying the optimized extraction protocols onthe various water samples results in graph 3.
In figure 3 we can clearly observe a corresponding high BEQ of endocrine activity (~17ng E2-eq./L)
coinciding with the CSO event in June 2015 at location Z9. Interestingly enough, the highest SPM content
at Z5 does not yield a high BEQ suggesting that most endocrine agonists enter the Zenne river after Z5
and before Z9. Within the various Zenne locations there is also a clear trend of increasing BEQ level
when moving from the upstream part (Z3) to the most downstream sampling point (Z11), see also figure
3. BEQ levels are highest in the hospital raw waste (average 137ng E2-eq./L), followed by the wastewater
received by the treatment plants South (av. 88ng E2-eq./L influent and 1.5ng E2-eq./L as effluent) andNorth (averaging, respectively, 40 and 2.5ng E2-eq./L for in- and effluent). The latter show, respectively,removal capacities of approx. 97.5% and 93.6% resulting in a disposal of treated water that is similar in
BEQ level as Zenne River water from upstream locations (Z3).
Comparison of the values observed during this study revealed that levels in the river waters werehigh compared to other literature where values ranged from 0.2-0.5ng E2-eq/L for surface waters and0.4-1.0ng E2-eq./L for municipal effluents [5]. However, ranges have also been reported from 0.53 to
17.9ng EEQ/L [14] similar to our findings, with 2/3 of the samples remaining below 0.5ng E2-eq./Lindicating the higher and constant load present in the Zenne River water samples during this study (allsamples ≥0.7ng E2-eq./L). Under the WFD the Environmental Quality standards have been proposed
at 0.4ng/L for E2 and 0.035ng/L for EE2 following chemical specific analysis [3]. Although BEQs arepresented here, which take into account all endocrine active agonists along with their possible mixtureeffects, their levels can be considered a concern [15]. For instance, PNECs have been established atlevels as low as 1ng E2/L (molecule specific) [15]. Given that our results are in vitro and endpointspecific, with no clear-cut extrapolation to in vivo effects (pharmacokinetics, in vivo metabolism, …)
and keeping in mind that BEQ values are presented here as E2 equivalents (without knowing the exact
contribution of pure E2 to the BEQ result, if any), future work will thus focus on carrying out compound
specific analyses to determine concentrations of E2 and EE2 and identify as well as characterize otherestrogenic chemicals.
Acknowledgements
The CALUX bioassay BG1Luc4E2 cell line was developed with funding from the National Instituteof Environmental Health Sciences Superfund Research grant (ES04699) to M.S. Denison. The authorswish to extend their gratitude to Innoviris in funding this research by awarding a Prospective Researchfor Brussels Grant (Anticipate).
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