Endocrine Disrupting Chemicals (EDCs) and Pathogens
removal in an hybrid CW system for a tourist facility
wastewater treatment and reuse

F. Masi*, G. Conte*, L. Lepri**, T. Martellini** and M. Del Bubba** * IRIDRA S.r.l., Via Lorenzo il Magnifico 70, 50129, Florence, IT (E-mail: [email protected]; [email protected]) ** Dept. of Chemistry, University of Florence, Via della Lastruccia 3, 50019, Sesto Fiorentino, Florence, IT (E-mail: [email protected]; [email protected]) Abstract
Within the frame of the “SWAMP” project (a demonstration project co-financed by the European
Community) an hybrid constructed wetland (CW) system was realized for the treatment of wastewater
produced by a medium-size hotel located in Florence, Italy. The system consisted of a 70 m3 primary
treatment by Imhoff tanks, a 160 m2 HF and a 180 m2 VF. The results obtained in the first year of
operation regarding to the hygienic indicator bacteria (total coliforms, faecal coliforms, faecal
streptococci and Escherichia Coli) and some selected organic compounds (Phthalates, PAHs and
Estrogens) were reported. In terms of overall performances the following mean removal rates were
obtained: COD 94%, BOD5 95%, TSS 90%. The CW system showed a satisfactory functioning of the
treatment process, especially related to the sedimentation and filtration processes and to organic matter
trapping and degradation inside the reed beds. As regard the EDCs a clear water quality increase
during the purification process was observd. PAHs were found below 0.5 µg/l in the inlet and at
significantly lower amounts in the outlet of the system. Total inlet phthalates concentration was
included in the range 100 to 300 µg/l and this class of compounds showed a certain persistence even in
the outlet (10-200 µg/l) due to HDPE liner release. Among the other compounds, 17-α-estradiol and
ethynyl-estradiol were revealed in the inlet at concentration below 10 µg/l, while were absent in the
Hybrid systems, Endocrine disruptors, Sustainable water management, Constructed wetlands, Reed
beds, Wastewater reuse
Chemical substances that can interfere with the normal functioning of the endocrine system have
been termed Endocrine Disrupting Chemicals (EDCs) (Keith et al., 1997). The full list of EDCs
includes a large range of anthropogenic and natural organic compounds, such as phthalates,
pesticides, polychlorinated biphenyls (PCBs), dioxins, polyciclic aromatic hydrocarbons (PAHs),
alkylphenols, bisphenols and steroid estrogens (Birkett and Lester, 2003). Several of these
substances have been released in increasing amounts in the environment since decades, and, due to
their low degradation rate, a significant increase of their background concentrations have been
observed in the different environmental compartments (Skakkebaeck et al, 2000, Tyler et al 1998).
In wastewater, EDCs, have a variety of source. A number of EDC classes (phthalates, pesticides,
PCBs and bisphenols) are industrial products, worldwide used for several applications and are
therefore ubiquitous pollutants (Staples et al, 1997, Kupfer, 1975, Safe S.H., 1994, Chen et al,
2002). Other kinds of EDCs compounds (dioxins and PAHs) are not commercial products, but are
formed as by-products of various industrial and combustion processes; they are transported from
atmosphere to soil and water bodies by the atmospheric runoff or deposited on the soil during the
dry period and then go through the water cycle by land runoff (Birkett and Lester, 2003).
Alkylphenols are metabolites of their ethoxylate precursors, which are non-ionic surfactants used in
many industrial, commercial and household functions (Del Bubba and Lepri, 2002). The presence of steroid estrogens in wastewater mainly arises from direct women excretion, in particular from pregnant females and women using oral contraception or hormone replacement therapies (Arcand-Hoy et al, 1998; Andrews, 1995). Several studies have shown that EDCs are present in the effluents from domestic and industrial conventional sewage treatment plants (Birkett and Lester, 2003) and this evidence is of potential concern about their reuse for non industrial applications, such as irrigation of crops and aquaculture, since EDCs could represent a contamination source of food chain. The behaviour of some EDCs has been studied in pilot constructed wetlands pointing out that reed beds are effective in the removal of phthalates and alkylphenol ethoxylates (Del Bubba et al., 1998; Del Bubba and Lepri, 2002). Another crucial parameter in order to assess the suitability of effluent wastewater to be reused is the occurrence of pathogens, which presence is usually evaluated by the analysis of the indicators total coliforms (TC), faecal coliforms (FC) and faecal streptococci (FS). Such microorganisms are commonly removed at high percentages in conventional sewage treatment plants and in constructed wetlands (Karpiscak et al., 2001; Arias et al., 2003), but the evaluation of residual concentrations is of strong importance for establishing the performances of the treatment plant and the final destination of the effluent wastewater. For these reasons, the understanding of removal processes and fate of EDCs and pathogens during wastewater treatment, may facilitate controlling or limiting exposure of both humans and environment to these compounds and create more solid basis for the choose of different Sustainable Water Management approaches. This paper presents the removal data of some EDCs and of TC, FC, FS and Escherichia Coli (which is required by the Italian national law about water reuse, D.Lgs. 185/2003) for an hybrid reed bed system located at an Hotel in Florence (Italy). This research was realized under the EC FP5 project SWAMP (for more details about the project see the website MATERIAL AND METHODS

Description of the CW system
The reed bed is composed by a first stage horizontal submerged flow (HF) and a second stage
vertical flow (VF) (see Fig. 1). In Table 1 are reported the main features of the reed bed system,
including the measured hydraulic loading rate (HLR) and organic loading rate (OLR) during the
monitoring period. In fact, an important characteristic of this treatment plant is the tourist
fluctuation, which involves a high variability of the daily flow and, consequently, of the loading
rate. A pump station located after the Imhoff tanks regulates the loading inside the HF bed by a
floating valve and so proportionally to the raw wastewater production. The effluent from the HF
bed is divided by a partition well into two independent siphons which feed the VF filter in an
alternate way. The waterproofing of the bed has been realized by an HDPE geo-membrane.
Fig. 1 - Schematic representation of the hybrid constructed wetland at the Relais Certosa Hotel. Table 1 – Main features of the Relais Certosa Hotel wastewater treatment facility Parameter
HF: mean 0.17 min 0.11 max 0.23 VF: mean 0.15 min 0.10 max 0.21
Sampling and analyses
As regards the ECDs analyses, grab samples were collected in glass bottles previously washed with
hot chromic mixture and repeatedly rinsed with ultra pure water; collection of samples for sanitary
indicators was carried out in glass bottles previously sterilized by autoclave treatment.
The sampling took place monthly from September 2003 to February 2004 (six samples), at the inlet
of the HF stage (after the Imhoff tank) and at the outlet of both the HF and VF stages. Inlet grab
samples were collected three days before the outlet ones, according to the estimated hydraulic
retention time (HRT) of the HF bed. Sampling campaigns were carried out when rain did not occur
within three days before the collection of the inlet and the sampling of the outlet samples, so as to
avoid dilution effects and HRT shortening, even if the rainwater is managed by a separated sewage
Analyses of ECDs were performed, separately for dissolved and particulate phases, after filtration
of the samples. In order to determine the concentration of particulate matter, filtration of each
sample was carried out on two aliquots of wastewater. One of the two filters was used for the
extraction of EDCs, while the other was dried in oven at 105°C for one hour and finally weighed.
Water phase, previously modified for pH and polarity by adding hydrochloric acid, sodium chloride
and methanol, was extracted by a triple liquid/liquid treatment with methylene chloride, evaporated
to 200 µl and analysed by a GC/MS Varian, model Saturn 4D in “micro selected ion storage (micro
SIS)” way. For phenols and estrogens a derivatization with N,O-bis-(trimethylsilyl)-
trifluoroacetamide (BSTFA) needed, in order to enhance the GC/MS response (Del Bubba et al.,
Particulate samples were extracted according to the method of Desideri et al. (1987), significantly
modified in order to obtain satisfactory recovery percentages (60-97%) for all the investigated
compounds (Del Bubba et al., 2004). Instrumental analysis of the extracted compounds was
performed as described above.
The bacteriological indicators TC, FC, FS and Escherichia Coli were evaluated according to the
protocols for membrane filter procedures of Standard Methods for the Examination of Water and
Wastewater (1995).
Removal of indicator bacteria

The average removals of the four analysed hygienic indicators were in the range 99.93-99.99%,
showing a very high efficiency of the system to remove pathogens (Tables 2-5). During the passage
through the HF Reed Bed the bacteria were reduced 2.9-3.2 log units, whereas the reduction in the
second stage VF bed was 0.7-1.2 log units.
Table 2 - Changes in Total Coliform density (cfu/100ml) through the CW system
Aug03 Sep03 Oct03 Nov03 Dec03 Jan04 Feb04 Mar04 1x107 3x107 2x107 1x107 4x106 7x106 7x105 4x106 2x106 8 x105 3x106 Table 3 - Changes in Fecal Coliform density (cfu/100ml) through the CW system Aug03 Sep03 Oct03 Nov03 Dec03 Jan04 Feb04 Mar04 8x106 3x107 7x106 8x106 2x106 3x106 5x105 7x105 1x106 4x105 9x105 Table 4 - Changes in Faecal Streptococci density (cfu/100ml) through the CW system Jul03 Aug03 Sep03 Oct03 Nov03 Dec03 Jan04 Feb04 Mar04 4x106 1x106 2x105 4x105 4x105 3x105 4x105 4x105 3x105 Table 5 – Changes in Escherichia Coli density (cfu/100ml) through the CW system Aug03 Sep03 Oct03 Nov03 Dec03 Jan04 Feb04 Mar04 6x106 3x107 7x106 6x106 2x106 2x106 4x105 6x105 1x106 4 x105 5x105 From such data, the following considerations can be highlighted: the reed population establishment inside HF and VF beds during July-August 2003 seems to have increased the pathogens removal in comparison to the first operating months; since October 2003 the removal rates increased, despite the different loading rates and the strong temperature changes during the seasons (water temp. range: 7-25 °C); from the same month the treated wastewater fulfilled the Italian regulation limits for reuse as regard the pathogens indicator E. Coli (80 percentile equal to 50 cfu/100ml and maximum admitted value equal to 200 cfu/100ml).
Removal of EDCs
The investigated organic compounds and their endocrine disrupting potencies (EDP), expressed as
17-ß-estradiol equivalent, are reported in Table 6.

Table 6 – Organic compounds investigated in the present work and their endocrine disrupting potencies (EDP); n.a. = quantitative data or magnitude order not available Compound
Among these compounds, estriol, butylbenzylphthalate, phenols and the most part of PAHs were never detected in the analysed samples. As regards PAHs, the compounds found in the inlet samples of the CW system were only Naphthalene, Phenanthrene, Fluoranthene and Pyrene, which concentrations for water and particulate phase ranged from 15 to 180 ng/L and from 2 to 7 mg/Kg, respectively. This clearly indicated a strong enrichment of PAHs in the particulate phase, according to their high value of log KO/W (Naphthalene 3.45; Phenanthrene 4.46; Fluoranthene 6,12; Pyrene 6.04). These compounds were below the detection limits (7-9 ng/L) in the outlet particulate matter, while the outlet concentrations for the dissolved phase ranged from 13 to 22 ng/L, giving rise to a removal efficiencies of about 60-70%. Steroid estrogens were detected in two inlet samples in the range 164 - 259 ng/L and 2.3 - 2.9 mg/Kg for water and suspended matter, respectively. These data pointed out that also for estrogenic compounds the adsorption onto particulate material is high, according to the log KO/W values, included in the range 3.43 (estrone) – 4.67 (mestranol). All the estrogens were below detection limits (15 ng/L) in dissolved and particulate phase of outlet samples of the HF stage, pointing out the high efficiency of this plant in the estrogen removal. Phthalates, with the exception of the butylbenzyl ester, were determined in all the inlet samples and were, generally, the most abundant compounds. For the dissolved phase concentrations changed in the ranges 151-3788, 43-6134 and 40-2480 ng/L, for diethyl, di-n-butyl and bis-2-ethylhexylphthalate, respectively. The concentrations found in the particulate phase were affected by a less variation and were in the ranges 33-152, 49-194 and 209-4566 ng/L. According to the different values of log KO/W for the above-mentioned phthalates (ranging from 1,46 for diethyl to 13,1 for bis-2-ethylhexyl), the enrichment factors increased, going from diethylphthalate to bis-2-ethylhexylphthalate. The trend of phthalate along the CW system was different from those of the other classes of EDCs as shown in Figure 2 and 3 for dissolved and particulate phase, respectively. As shown by figures, diethyl and di-n-butyl-phthalate were highly removed during the treatment, while for bis-2-ethylhexylphthalate an opposite trend was observed. This trend was found in all the analysed samples suggesting that a source of this compound could be present inside the plant. In order to explain this unexpected behavior a little portion of the HDPE geo-membrane (50 cm2) was placed in water (HPLC grade) under stirring for three days (the design HRT). The results of this test pointed out that the membrane released all the three phthalates. However the compound released at highest concentration was bis-2-ethylhexylphthalate, which accounted for more than 70% of the total phthalate release. These findings explain the different observed trend among phthalates. Fig. 2 – Concentration of diethyl, di-n-butyl and bis-2-ethylhexylphthalate in the dissolved phase of the sample collected on December 2004. Fig. 3 - Concentration of diethyl, di-n-butyl and bis-2-ethylhexylphthalate in the particulate phase of the sample collected on December 2004.

The hybrid CW system, realized for the treatment of wastewater produced by a medium-size hotel,
was suitable for the removal of pathogens and of ECDs since eight months from its conctruction,
and the outlet water was able to be reused according to Italian regulations. Among ECDs trace
amounts of estrogens (17-α-estradiol and ethynyl-estradiol), PAHs (Naphthalene, Phenanthrene,
Fluoranthene and Pyrene) and phthalates (diethyl, di-n-butyl and bis-2-ethylhexylphthalate) were
found in inlet wastewater. All of these compounds were removed at high percentages (up to 100%
for estrogens), with the only exception of bis-2-ethylhexylphthalate, which was released by the
HDPE liner. Therefore the use of geo-membrane with no ECDs release it is of paramount
importance, especially for the reuse of the final effluent.
This study was partially funded by the European Commission under the Fifth Framework
Programme and contributing to the implementation of the Key Action “Sustainable Management
and Quality of Water” within the Energy, Environment ans Sustainable Development Programme,
project “SWAMP” n° EVK1-CT-2000-00071. The authors are solely responsible for the content of
this paper that does not represent the opinion of the Community. The European Community isn’t
responsible for any use that might be made of data appearing therein.
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