Ecological Engineering 21 (2003) 233–247

Feasibility of using ornamental plants (Zantedeschia aethiopica)

in subsurface flow treatment wetlands to remove nitrogen,
chemical oxygen demand and nonylphenol ethoxylate
surfactants—a laboratory-scale study
Marco A. Belmont∗ , Chris D. Metcalfe
Environmental and Resource Studies Program, Trent University, Peterborough, Ont., Canada K9J 7B8

Received 14 March 2003; received in revised form 18 September 2003; accepted 13 October 2003

Abstract

A lab-scale subsurface flow wetland (SSFW) was studied to assess the effectiveness of using calla lily (Zantedeschia aethiopica)
in SSFW to remove nitrate, ammonium, chemical oxygen demand (COD), and nonylphenol ethoxylate (NPEO) surfactants.
Reduction of inorganic nitrogen and COD from wastewater is one of the most important objectives in any water treatment
process and is usually used to evaluate the performance of the treatment system. NPEO surfactants are of interest because
they are contaminants found in sewage effluents at ppm concentrations that have estrogenic potential. It is very important to
remove N, COD, and NPEO from wastewater for protection of aquatic life. The removal performances of these compounds in
cells planted with calla lily were compared with unplanted cells. Nitrate, ammonium, total Kjeldahl nitrogen (TKN), dissolved
oxygen (DO), redox potential (Eh), hydrogen potential (pH), and COD were quantified in the influent and effluent to evaluate
the performance of the wetland in the wastewater treatment. In a separate experiment, removal rates of NPEO surfactants were
determined. High removal rates of nitrogen, COD and NPEO were observed in the SSFW. Comparisons between the planted
and unplanted treatments indicated that Z. aethiopica significantly influenced the removal rate of nitrogen but not the removal
rate of COD or NPEO surfactants.
© 2003 Elsevier B.V. All rights reserved.

Keywords: Nonylphenol; Nonylphenol ethoxylates; Non-ionic surfactants; Treatment wetlands; Subsurface flow wetlands; Ornamental plants;
Wastewater

1. Introduction

Constructed wetlands are potentially a low-cost

∗ Corresponding author. Present address: Soil and Water Sci-
ence Department, Institute of Food and Agricultural Science,
University of Florida, P.O. Box 110510, 106 Newell Hall,
Gainesville, FL 32611, USA. Tel.: +1-352-392-1804x316; fax:
+1-352-392-3399.
E-mail address: mbelmont@ifas.ufl.edu (M.A. Belmont).
solution for treating domestic and industrial wastew-
ater in developing countries, such as Mexico
(Denny, 1997; Kivaisi, 2001). However, treatment
of wastewater is not a high priority in small rural
communities in most developing countries unless the

0925-8574/$ – see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.ecoleng.2003.10.003
234 M.A. Belmont, C.D. Metcalfe / Ecological Engineering 21 (2003) 233–247
communities can derive economic benefit from the
water resources that are created by the treatment pro-
cess. As part of studies directed at improving water
quality in central Mexico, the feasibility of using
ornamental flowers for treatment of domestic wastew-
ater was assessed using a laboratory-scale subsurface
flow wetland. The wetland was planted with calla lily
(Zantedeschia aethiopica), which is a flower with a
high market value in Mexico (Auman, 1996). Flori-
culture activities in constructed wetlands could pro-
vide the economic benefits necessary in encouraging
small communities to maintain wastewater treatment
systems.
It is feasible to treat domestic wastewater in small
rural communities using constructed wetlands. These
systems are especially valuable for on-site wastewa-
ter treatment in developing countries because they in-
volve simple technology and the costs of construction
and operation are low (Denny, 1997). The production
of vegetational biomass in treatment wetlands can pro-
vide economic returns to communities upon harvest.
These economic benefits can be realized through pro-
duction of “bio-gas”, animal feed, compost, and fiber
for paper making (Lakshman, 1987). There have been
no reports on the use of ornamental plants, such as
flowers, in constructed treatment wetlands, despite the
fact that these plants can be harvested and sold for
significant economic return.
Most of the research on constructed wetlands has
been conducted in northern countries. As a result, the
plants most studied for treatment purposes are cat-
tail (Typha spp.), bulrush (Scirpus lacustris) and reeds
(Phragmitis australis), as these plants can withstand
cold winters (Denny, 1997). It is known that ornamen-
tal plants such as canna lily (Canna flaccida), calla lily
(Z. aethiopica), elephant ear (Colocasia esculenta),
ginger lily (Hedychium coronarium), and yellow iris
(Iris pseudacorus) can be used in rock/plant filters to
treat septic tank effluents (Wolverton, 1990). However,
there is little information about the efficacy of orna-
mental plants for use in treatment wetlands. The fact
that these ornamental plants cannot survive the cold
winters in northern countries may be one reason why
they have had limited use in constructed wetlands.
Since most developing countries are located in tropical
and subtropical regions and have limited resources to
install conventional wastewater treatment systems, the
use of ornamental plants in treatment wetlands should
be explored. Floriculture opportunities would also pro-
vide economic benefits to communities, in addition to
the environmental benefit of treating the wastewater.
In addition to evaluating the use of ornamental
plants in subsurface treatment wetlands, this research
was also conducted to study the removal of Alkylphe-
nol ethoxylates (APEO), which are endocrine dis-
ruptors (Routledge and Sumpter, 1997; White et al.,
1994; Gray and Metcalfe, 1997; Metcalfe et al., 2001)
and have been found in wastewater treatment plants
effluents (Bennett and Metcalfe, 2000; Bennie et al.,
1998; Solé et al., 2000; Stephanou and Giger, 1982).
The fate of these organic pollutants in treatment
wetlands has not been studied.
APEO are non-ionic surfactants commonly used
in cleaning products and in a variety of industrial
processes (Tolls et al., 1994). APEO are a mix-
ture of polyethoxylated alkylphenols, predominantly
para-substituted, which are used in the manufactur-
ing of paints, detergents, inks, and pesticides and
are constituents of household cleaners and detergents
(Weinberger and Greenhalg, 1984). The most widely
used alkylphenol ethoxylates are the nonylphenol
ethoxylates (NPEO), although derivatives of octylphe-
nol are also used in some products. These surfactants
have received attention recently because microbial
degradation of these compounds produces interme-
diates that have estrogenic activity (Routledge and
Sumpter, 1997; White et al., 1994; Gray and Metcalfe,
1997; Metcalfe et al., 2001). Hydrolytic shortening
of the polyethoxylate chain of NPEO is generally
favored under anaerobic conditions, leading to the
formation of lower oligomers, such as those having
two (NP2EO) and one (NP1EO) ethoxy units and
ultimately to the completely de-ethoxylated product,
nonylphenol (NP) (Giger et al., 1984; Ahel et al.,
1994b). Furthermore, NP2EO and NP1EO can be
oxidized under aerobic conditions to nonylphenol car-
boxylic acids, NP2EC and NP1EC (Ahel et al., 1986,
1994a).
NPEO has been found in the effluents of domestic
waste water treatment plants (WWTP) (Bennett and
Metcalfe, 2000; Bennie et al., 1998; Solé et al., 2000;
Stephanou and Giger, 1982). It was estimated that
NPEO could represent between 4 and 10% of the total
dissolved organic carbon entering a waste water treat-
ment plant (WWTP) (Ahel et al., 1994a). The origi-
nal NPEO surfactants are not particularly estrogenic;
 M.A. Belmont, C.D. Metcalfe / Ecological Engineering 21 (2003) 233–247
however, partial breakdown in the sewer and treatment
works yields some estrogenic intermediates (Jobling
et al., 2002; Metcalfe et al., 2001). There have been
studies on the fate of NPEO in WWTP (Johnson and
Sumpter, 2001; Ahel et al., 1994b; Di Corcia et al.,
1994), but to our knowledge, there are no studies on
the fate of these estrogenic compounds in treatment
wetlands. Since the use of treatment wetlands have
become more popular for wastewater treatment and
WWTP effluent polishing, it is important to investigate
the fate of NPEO in these treatment systems due to the
possible environment impacts of these compounds.
This research was conducted to evaluate the effec-
tiveness of using Z. Aethiopica in subsurface flow wet-
lands to remove nitrogen, COD and NPEO surfactants
from simulated domestic wastewater.
2. Materials and methods
2.1. Lab-scale wetland
A lab-scale subsurface flow wetland was built in
a greenhouse at Trent University, Peterborough, On-
tario, Canada. The system consisted of six cells; each
38 cm wide and 240 cm long. The substrate consisted
of a 30-cm layer of crushed rock (3–5 cm diameter).
The porosity of the media was φ = 0.35. The water
level was kept at 5 cm below the gravel surface and
the volume of water in each cell was 80 l. Including
the water in the elevated header tank, the total volume
involved in the experiment was approximately 1000 l.
Fig. 1. Lab-scale subsurface flow wetland (SSFW).
235
In two different phases of the experiment, flow rates
were adjusted to 27.8 ml/min for a hydraulic retention
time (HRT) of 2 days, and to 55.5 ml/min for a HRT
of 1 day.
Four cells were planted with calla lily and two cells
were left unplanted to be used as reference cells. Sim-
ulated wastewater was stored in an elevated header
tank (Fig. 1). This simulated wastewater consisted
of water from the Otonabee river in Peterborough,
Ontario, spiked with fertilizer and tannic acid, as
described below. All cells were fed with the same
simulated wastewater at the same flow rate. The efflu-
ent was collected and pumped back into the elevated
tank. Nutrients were added periodically to the tank
to keep the characteristics of the simulated sewage
as constant as possible. The simulated wastewater
was prepared, to fall within the range of water qual-
ity parameters for “weak” sewage in North America
(Table 1), as defined by Metcalf and Eddy (1991).
2.2. Plants
Each calla lily cell was planted with 22 plants, on
15 cm centers (distance between them). The plants
were purchased from a nursery company based in
British Columbia and were 8 cm tall when planted.
The system was maintained at a temperature range of
14–20 ◦ C and 16 h of natural/artificial light per day.
After planting, nutrients were added to the water in
the same composition recommended for hydroponics
to allow the plants to root and grow. The composition
of this nutrient solution is indicated in Table 2. During
the experiment, the flowers produced were harvested.
236 M.A. Belmont, C.D. Metcalfe / Ecological Engineering 21 (2003) 233–247

Table 1
Typical range of water quality parameters in North American domestic wastewater and range of parameters in the lab-scale experimental
wetland influent
Parameter Units Sewage in North Americaa Experimental wetland

Weak Medium Strong

Total suspended solids (TSS) mg/l 100 220 350 6–15

Ammonium (NH4 + ) mg/l 12 25 50 19–27
Nitrate (NO3 − ) mg/l 0 0 0 4–7
Nitrite (NO2 − ) mg/l 0 0 0 0.1–0.2
Chemical oxygen demand (COD) mg/l 250 500 1000 62–90
Dissolved oxygen (DO) mg/l – – – 1.5–1.9
Hydrogen potential (pH) pH units – – – 6.9–8.1
Redox potential (Eh) mV – – – 134–190
Orto phosphate (PO4 3− ) mg/l – – – 25–75
Sulfate (SO4 2− ) mg/l 20 30 50 600–620
Chloride (Cl− ) mg/l 30 50 100 43–57
a Metcalf and Eddy (1991).

2.3. Additions of nutrients and NPEO all, the influent water was similar in composition to a
weak domestic wastewater.
Once the plants were well established (i.e., 120 days In a separate experiment, a commercial mixture of
later), 250 g of lawn fertilizer and 250 g of tannic acid NPEO surfactant was added to the elevated header
were added to increase ammonium, nitrate and COD tank, along with nutrients. NP(1–3)EO in the form of
concentrations, because these were the parameters of 52 g of Igepal CO-210 (Aldrich, Cat. No. 26636-32-8)
interest in this study. The fertilizer used was the com- was added to the header tank at the beginning of the
mercial formula 26-4-4 (26% N, 4% P, 4% K2 O, 3.1% experiment. This product is a commercial mixture that
sulfur, and 15% organic matter). The first addition was contains mainly NP1EO, NP2EO and NP3EO. Igepal
completed on 14 April 2000. Based on the results of was then added at a rate of 2 g per week to the header
the analyses of the influent, subsequent additions of tank to maintain the concentrations of NPEO in the
fertilizer and tannic acid were done every 5 or 6 days, influent slightly higher to the concentrations observed
and the amounts added were between 100 and 250 g in wastewater treatment plants effluents.
each, as needed to maintain the desired levels of COD
and N. The characteristics of the influent water in the 2.4. Experiments
lab-scale experimental wetland are shown in Table 1,
along with the typical values for these parameters in Experiments were performed to determine the ef-
domestic wastewater (Metcalf and Eddy, 1991). Over- fects of treatment on nitrogen and COD removal at
two different hydraulic retention times (1 and 2 days).
Based on the results of these experiments, another ex-
Table 2 periment was performed with a hydraulic retention
Nutrients added after planting to help establish the calla lily plants time of 1 day to study the removal and transformation
8 December 17 January 14 March
of nonylphenol ethoxylates in the system:
1999 (g) 2000 (g) 2000 (g)
1. One experiment at HRT = 2 days (27 June 2000
CaNO3 800 500 260
to 17 August 2000). The influent and effluent were
KNO3 500 – 150
K2 SO4 225 567 500 monitored for pH, Eh, DO, COD, ammonium, and
KH2 PO4 210 275 133 nitrate. The flow rate was adjusted to 27.8 ml/min
MgSO4 400 500 246 for an HRT = 2 days.
Trace elements 26 26.2 13 2. Two experiments at HRT = 1 day (11 February
The total volume of the water was 1000 l. 2000 to 11 May 2000, and 26 September 2000 to
 M.A. Belmont, C.D. Metcalfe / Ecological Engineering 21 (2003) 233–247 237

2 November 2000). The influent and effluent were Eh and pH, were measured with Orion electrodes,
monitored for pH, Eh, DO, COD, total Kjeldhal model numbers 9778BN and 9107BN, respectively.
nitrogen (TKN), ammonium, and nitrate. The flow The meter was calibrated using buffers of borax (pH
rate was adjusted to 55.5 ml/min for an HRT = 1 9.2) and phthalates (pH 4.0) for pH, and Zo Bell’s
day. solution for Eh (APHA, 1998). Total suspended solids
3. One experiment at HRT = 1 day with addition of (TSS) were quantified by filtering water samples
NP(1–3)EO (15 April 2001 to 22 August 2001). through pre-weighed glass–fiber filters (Whatman
The influent and effluent were monitored for pH, GF/C, 42-mm diameter, 1.2-␮m pore size) and dry-
Eh, COD, ammonium, and nitrate. The flow rate ing at 110 ◦ C. The volume of water filtered was ∼4 l.
was adjusted to 55.5 ml/min which corresponds to Dried filters were weighed to four decimal places.
an HRT of 1 day. TKN was measured by a contract laboratory (Lake-
field Research, Lakefield, Ontario, Canada) using the
Water samples were collected three times a week Micro Kjeldhal method (APHA, 1998).
during the experiments. Eight samples were taken each Chemical oxygen demand (COD) was analyzed
time: two samples from the influent, and one from the by the closed reflux photometric method using com-
effluent of each of the six cells. mercial kits (BioScience Inc., Cat. No. 174-318).
The standard curves were constructed using stan-
2.5. Water analyses dards of potassium biphthalate of 50, 125, 250 and
500 mgCOD /l.
Water monitoring was conducted according to tech- Three sets of samples were analyzed for phosphate,
niques described in the Standard Methods for Water sulfate and chloride in the influent. Samples were
and Wastewater Analysis (APHA, 1998) and a number analyzed by ion chromatography using a DIONEX
of water quality parameters were measured (Table 3). chromatograph. These analyses were done periodi-
Concentrations of ammonium, nitrate and nitrite cally over the length of the experiment to assure the
were measured using ORION ion-selective electrodes content of chloride, sulfate and phosphate in the sim-
(model number 9512BN, 9707BN, and 9746BN, re- ulated wastewater was within the acceptable range of
spectively). The calibration curves were created using values.
standards of ammonium chloride, sodium nitrate,
and sodium nitrite, respectively (1, 10, and 100 ppm 2.6. Analysis of nonylphenol (NP) and nonylphenol
for ammonium and nitrate, and 0.1, 1.0 and 10 ppm ethoxylate surfactants (NPEO)
for nitrite). Dissolved oxygen was quantified using a
YSI oxymeter. The instrument was calibrated against 2.6.1. Solid phase extraction
saturated air. NP and NPEOs were extracted by solid phase ex-
traction (SPE) as described by Ferguson et al. (2000).
Water samples (100 ml) were acidified to pH < 2
Table 3 using trace grade sulfuric acid and then, distilled in
Analytical techniques used for water analyses
glass (DIG) methanol was added at 0.5 ml/100 ml
Parameter Method water to aid in SPE extraction. Plastic SPE tubes
pH Electrode (6 ml, Supelco) were packed by first inserting a
Temperature Thermometer PTFE frit to the bottom and then adding 1.0 g of
Eh Pt electrode C18 packing (Bondesil, 40 ␮m, Varian), followed by
Total suspended solids Glass fiber filter dried at 110 ◦ C
another frit as a cap. Prior to sample addition, the
Ammonia Ion selective electrode
Nitrate Ion selective electrode
columns were pre-rinsed with 10 ml of DIG acetone,
Nitrite Ion selective electrode 10 ml of methanol, and then 10 ml of Milli-Q wa-
Total Kjeldhal nitrogen Micro TKN ter, adjusted to pH 2 with trace grade sulfuric acid.
Chemical oxygen demand Closed reflux method Samples were passed through the columns at a rate
(photometric) of 10 ml/min. Once the samples were extracted, their
Dissolved oxygen Membrane electrode
respective containers were rinsed three times with
238 M.A. Belmont, C.D. Metcalfe / Ecological Engineering 21 (2003) 233–247
acidified Milli-Q water to ensure complete extraction
of the entire sample. Vacuum was applied for 90 min
after rinsing to air-dry the tubes. To elute the samples,
two 3-ml aliquots of DIG acetone were added to the
SPE tubes and the eluent was collected in screw-top
vials. For each aliquot, the solvent soaked the sorbent
bed for 10 min. Once eluted, samples were taken to
dryness using a Savant vacuum system and then recon-
stituted by adding 0.25 l DIG methanol, 0.25 ml iso-
propyl alcohol, and 4.5 ml DIG hexane, with vortex-
ing between each addition. The reconstituted volume
was 5 ml.
2.6.2. Chromatographic analysis
Nonylphenol and nonylphenol ethoxylates in
the effluent of the wetland were quantified by
high-performance liquid chromatography with
electrospray ionization and mass spectrometry
(LC–ESI–MS). The concentrations of these com-
pounds in the influent, which were at concentrations
higher than in the effluent, were determined by
high-performance liquid chromatography and diode
array detection (HPLC–DAD). The analytical sys-
tem used was a Hewlett-Packard 1100 series HPLC,
equipped with a diode array detector and a mass
selective detector (MSD); a single quadrupole mass
filter. Chromatographic separation was achieved with
a normal phase column (3 ␮m HYPERSIL APS,
4.6 mm × 150 mm). Two mobile phases were em-
ployed: mobile phase A consisted of 98% HPLC grade
hexane and 2% distilled-in-glass isopropyl alcohol;
mobile phase B consisted of 90% distilled-in-glass
isopropyl alcohol and 10% distilled, deionized water.
The flow rate of mobile phase through the column
was 0.40 ml/min. For analysis of influent samples by
HPLC–DAD, the detector was set to monitor at a
wavelength of 227 nm, with a 16-nm slit width. This
wavelength allowed for approximate detection limits
of 30 ng/ml for NP and NPEO analytes in the sample.
NP and NPEOs were analyzed by LC–ESI–MS af-
ter post-column addition to the mobile phase of 90%
2-propanol and 10% water, made up to approximately
20 ␮M sodium acetate so as to produce (almost exclu-
sively) the Na+ ion adduct at m/z [M + 23]+ , where
M is the molecular mass of the un-ionized ethoxylate.
The post-column addition flow rates were 0.10 ml/min
for positive ion detection of NPEOs and 0.30 ml/min
for negative ion detection of NP. The electrospray
source was operated with a nitrogen drying gas tem-
perature of 350 ◦ C and a nebulizer pressure of 35 psi
(nitrogen).
In order to achieve maximum sensitivity, two anal-
yses were performed for each sample: one for the de-
termination of NP (negative ion mode) and the other
for the determination of NP1EO to NP3EO (posi-
tive ion mode). Selected ion monitoring mode was
employed to determine the respective concentrations
of the analytes; NP—m/z 219 (negative ion mode),
NP1EO—m/z 243 (positive ion mode), NP2EO—m/z
287 (positive ion mode), NP3EO—m/z 331 (positive
ion mode). Detection limits varied to some degree, de-
pending on the condition of the electrospray source
and the background chemical noise, but were typically
of the order of 3 ng/ml for NP and 0.5 ng/ml for the
NPEOs.
An external calibration method was performed
for the quantification of the sample extracts. Multi-
ple point initial calibration standards were analyzed
to establish linearity. Calibration standards were
re-analyzed after every ten to twelve samples. A mean
response factor (RF) was obtained from each pair of
calibration standards and used to quantify samples
which fell between the two sets of calibration stan-
dards. The mean RF was used to calculate the concen-
trations of the compounds of interest. The analytical
standard was supplied by Huntsman Chemical Co.,
Austin, TX, USA, and consisted of a mixture of NP
and ethoxylates up to 18 ethoxy units. As mentioned
above, only NP and the monomers, dimers and trimers
were examined in this study, so these compounds
were quantitated against the standard according to
the percentage composition supplied by Huntsman
Chemical: NP 2.7%, NP1EO 2.9%, NP2EO 3.3%
and NP3EO 5.7%. Thus, a 1-ppm standard contained
27, 29, 33 and 57 ppb, respectively, of the four com-
pounds. Standards were run at varying total concen-
trations of 0, 1, 3, 5 and 10 ppm, depending on what
was required for the particular set of samples. For the
lower concentrations, a linear fit to the response curve
sufficed, with an R2 coefficient of 0.99–0.999 in most
of the cases. For the higher concentrations, the posi-
tive response became non-linear, and a quadratic fit
was required to get calibration curves of sufficiently
high correlation coefficient. All data were reprocessed
using Hewlett-Packard G1030A MS ChemStation
(DOS Series) software.
 M.A. Belmont, C.D. Metcalfe / Ecological Engineering 21 (2003) 233–247 239

3. Results and discussion
Calla lilies grew well on the crushed rock substrate.
The growth of the plants was fast. After 60 days of
planting, the height of the plants was 50 cm on aver-
age and the roots were about 25 cm long. At this time,
new shoots emerged above the crushed rock substrate.
The plants started flowering by 6 weeks after plant-
ing. It has been observed that coarse, angular rock is
not a good substrate for constructed wetlands since
it inhibits the development of the roots (Kadlec and
Knight, 1996). However, it was observed in this ex-
periment that the roots developed well and the plants
did reproduce without difficulty in the substrate. Only
two plants died from the original 88 that were planted.
As shown in Table 1, the influent had higher con-
centrations of sulfate than typical values for sewage.
This occurred because the fertilizer that was added to
the simulated sewage contained high concentrations
of sulfate. However, data in the literature indicate
that this parameter does not significantly affect the
development of plants (Kadlec and Knight, 1996).
Levels of TSS were kept much lower than typical
values (Table 1) to avoid solids that might clog lines
and interfere with the control of slow flow rates. Ac-
cording to Table 1, the ammonium concentration in
the influent (19–27 mg/l) corresponds to a medium
polluted wastewater (typically 25 mg/l) while COD
(62–90 mg/l) was lower than the typical COD values
for weak wastewater (250 mg/l). Overall, the influent
simulated a “weak” domestic wastewater (Table 1).
Table 4 presents a summary of the parameters mea-
sured in samples of influent and effluent exiting from
the cells with calla lily, and from reference cells. The
data for both experiments at HRT = 1 day were
pooled. These parameters are discussed in detail in the
following sections.
3.1. Chemical oxygen demand
The COD levels in the influent were highly variable,
especially in the first experiment (HRT = 2 days).
However, the reduction of COD in the wetland is clear.
The COD reduction at a 2-day HRT was higher than
with a 1-day HRT (Fig. 2), which was expected ac-
cording to the literature (Kadlec and Knight, 1996).
For the first treatment (HRT = 2 days), the ANOVA
performed on this data set showed that the COD re-
duction by the calla lily cells and reference cells were
not statistically different (P = 0.883). The difference
between the mean COD levels in the influent and the
effluent from the cells planted with calla lily was sta-
tistically significant (P < 0.001). Significant differ-
ence in COD levels was observed between the influent
and the effluent of the reference cells (P < 0.001).
Similar results were observed for the second treat-
ment (HRT = 1 day). The COD levels in the calla lily
cells and reference cells were not statistically different
(P = 0.535), while the COD of the effluent of both
treatments differed significantly from the COD of the
influent (P < 0.001 and P = 0.0013, respectively).
These results indicate that the presence of plants was
not an important element for COD removal in the wet-
land.
The percentage COD removal at 2 days of HRT was
35.1±3.9% for the calla lily cells and 36.1±5.9% for
the reference cells. For the treatment with HRT = 1
day, the removal was 18.4 ± 4.2% for the ornamental

Table 4
Parameters measured in the lab-scale wetland at hydrologic residence times (HRT) of 2 and 1 day, respectively
HRT = 2 days HRT = 1 day

Influent Effluent calla lily Effluent reference Influent Effluent calla lily Effluent reference

COD (mg/l) 86.9 ± 11.7 57.7 ± 4.0 58.2 ± 4.9 62.1 ± 5.2 52.9 ± 2.5 51.6 ± 3.0
NH4 + (mg/l) 19.8 ± 7.8 9.0 ± 3.8 18.1 ± 7.1 26.3 ± 7.3 13.8 ± 2.9 23.4 ± 4.4
Nitrate (mg/l) 3.8 ± 1.3 4.7 ± 1.1 5.7 ± 1.8 7.0 ± 1.3 15.8 ± 2.0 10.7 ± 2.3
Nitrite (mg/l) 0.15 ± 0.06 0.18 ± 0.04 0.21 ± 0.05 – – –
TKN (mg/l) – – – 19.9 ± 6.1 11.0 ± 2.5 18.5 ± 4.4
DO (mg/l) 1.5 ± 0.2 3.1 ± 0.2 3.6 ± 0.2 1.9 ± 0.2 2.5 ± 0.1 3.3 ± 0.2
Eh (mV) 178 ± 14 205 ±7 202 ±9 144 ± 20 215 ± 4 207 ± 6
pH 7.75 ± 0.1 7.3 ± 0.1 7.8 ± 0.1 7.6 ± 0.1 7.2 ± 0.0 7.7 ± 0.1
240 M.A. Belmont, C.D. Metcalfe / Ecological Engineering 21 (2003) 233–247

Fig. 2. Mean COD (±95% confidence intervals), in mg/l, measured in input water and in outputs from ornamental plant and reference
cells at 2 and 1 day of hydraulic retention time (HRT).

plant cells and 20.1 ± 6.0% for the reference cells.
These results show that there is a significant difference
in COD removal rates between 1 and 2 days of HRT,
which is consistent with the literature (Kadlec and
Knight, 1996).
3.2. Nitrogen
3.2.1. Ammonium
In the experiments with HRTs of 1 and 2 days, there
was a noticeable reduction of ammonium in the calla
lily cells, while there was no substantial reduction
in the reference cells (Fig. 3). For the first treatment
(HRT = 2 days), there was no significant difference
between ammonium concentrations in the influent
and the effluent from reference cells (P = 0.747).
There was a significant difference in ammonium con-
centration between the effluent from the cells planted
with calla lily and the influent (P = 0.007). The
ANOVA also showed that there was a significant
difference in ammonium concentration between the
effluent from the calla lily cells and the reference cells
(P = 0.017).
For the second treatment (HRT = 1 day), there were
similar results. There was no significant difference
between ammonium concentrations in the influent
and in the effluent of the reference cells (P = 0.511)
but there was a significant difference between ammo-
nium in the influent and the effluent from calla lily
cells (P < 0.001). Also, the ammonium concentra-
tions in the effluent from calla lily and reference cells
were statistically different (P < 0.001). These results
indicate that calla lily plants have a positive effect on
ammonium removal in the wetland. The plants were

Fig. 3. Mean ammonium concentration (±95% confidence intervals), in mg/l, measured in input water and in outputs from ornamental
plant and reference cells at 2 and 1 day of hydraulic retention time (HRT).
 M.A. Belmont, C.D. Metcalfe / Ecological Engineering 21 (2003) 233–247
Fig. 4. Mean nitrate concentration (±95% confidence intervals), in mg/l, measured in input water and in outputs from ornamental plant
and reference cells at 2 and 1 day of hydraulic retention time (HRT).
involved in the ammonium removal process under hy-
draulic retention time conditions of 1 and 2 days.
3.2.2. Nitrate
Fig. 4 shows the data on mean nitrate concentra-
tions and the 95% confidence intervals (α = 0.05).
The ANOVA for the first treatment (HRT = 2 days)
showed that there was no significant difference be-
tween nitrate concentrations in the influent and efflu-
ents (P = 0.264). However, for the second treatment
(HRT = 1 day), significant lower concentration of ni-
trate (P < 0.001) was observed in the influent than
in the effluents. For this latter experiment (HRT = 1
day), nitrate concentrations in the effluent from calla
lily and reference cells were significantly different
(P = 0.003). Nitrate concentrations in the effluent
from calla lily cells and influent were also statistically
different (P < 0.001), as were the data for influent
and effluent from reference cells (P = 0.005).
The data from the second treatment showed an in-
crease in nitrate concentrations in the wetland cells,
indicating good conditions for nitrification of ammo-
nium to nitrate. The calla lily cells performed more ef-
ficient nitrification than the reference cells. In contrast
to the experiment at HRT = 1 day, the experiment at
HRT = 2 days did not show evidence of nitrification
in the planted and reference cells. The opposite was
expected, i.e. more nitrification at HRT = 2 days than
at HRT = 1 day. The possible explanation is that there
was more dissolved oxygen (DO) available for nitrifi-
cation in the experiment at HRT = 1 day than in the
one at HRT = 2 days. This statement is supported by
the fact that COD was reduced more at HRT = 2 days
241
than at HRT = 1 day (Fig. 2). This smaller reduction
of COD at HRT = 1 day (due to the shorter HRT
and lower influent COD) left DO available for nitrifi-
cation of ammonium, with a subsequent elevation of
nitrate in the water. This also explains why the DO
in the calla lily effluents was lower at HRT = 1 day
(2.5±0.1 mg/l) than at HRT = 2 days (3.1±0.2 mg/l)
(Fig. 6). This difference in DO was statistically sig-
nificant (P < 0.001).
3.2.3. Nitrite
In experiment 1 (HRT = 2 days), nitrite was
detected in the system at very low concentrations
relative to concentrations of nitrate and ammonium:
0.15 ± 0.06 mg/l in the influent, 0.18 ± 0.04 mg/l in
the calla lily cells, and 0.21 ± 0.05 mg/l in the ref-
erence cells (Table 4). Because concentrations of ni-
trate and ammonium appeared to be more descriptive
of the nitrogen transformation in this artificial wet-
land system, nitrite was not measured in the second
experiment.
3.2.4. Total Kjeldahl nitrogen
TKN was monitored from 19 September to 2
November 2000, in the treatment with HRT = 1 day.
The average TKN concentrations and the confidence
intervals (α = 0.05) are presented in Fig. 5. There was
no significant difference in TKN between influent and
effluent from reference cells (P = 0.512) but there
was a significant difference between TKN in influent
and effluent from cells with calla lily (P = 0.012), as
well as significant differences in TKN in the effluent
from calla lily and reference cells (P = 0.0030).
242 M.A. Belmont, C.D. Metcalfe / Ecological Engineering 21 (2003) 233–247

Fig. 5. Mean total Kjeldahl nitrogen (±95% confidence intervals), in mg/l, measured in input water and in outputs from ornamental plant
and reference cells, and percentage of reduction in ornamental and reference cells, at 1 day of hydraulic retention time.

There was a significant difference between the per-
centage of TKN reduction observed (P < 0.001),
which were 45.6 ± 6.7% and −7.3 ± 13.7% for the
calla lily and reference cells, respectively (Fig. 5). It
is clear that the plants promoted TKN reduction. This
agrees with the observation that the planted cells were
removing ammonium while there was no significant
change in ammonium concentration in the reference
cells. However, it must be pointed out that TKN lev-
els were lower than ammonium levels in these studies
(Table 4). TKN analysis, which was done under con-
tract, may not have been accurate, although precision
appeared to be good. The data indicated that there was
total nitrogen removal in the system, but we cannot at-
tach much certainty to the absolute levels of removal.
3.3. Dissolved oxygen
Fig. 6 shows the results of DO measurements. The
DO in influent was similar during both experiments.
The same was observed for the effluent from the
reference cells. However, the effluent from calla lily
cells showed greater DO at HRT = 2 days than at
HRT = 1 day (3.1 ± 0.2 mg/l and 2.5 ± 0.1 mg/l,
respectively). This difference was statistically sig-
nificant (P < 0.001). These results are consistent
with the earlier observation of less COD reduction
at HRT = 1 day than at HRT = 2 days (Fig. 2) and
greater nitrification of ammonium in the calla lily
cells at an HRT of 1 day than at an HRT = 2 days
(Fig. 4). There was no noticeable difference in nitri-
fication in the reference cells at the different HRTs
(Fig. 4). Another factor that possibly influenced this
behavior was the fact that the water was in contact
with the roots of the plants for a longer period of
time, allowing for greater oxygenation of the water.
The DO levels in the reference cell effluents were
higher than in the effluent from the planted cells. This
may be explained by the fact that there was reduction
of ammonium in the planted cells, but none in the
 M.A. Belmont, C.D. Metcalfe / Ecological Engineering 21 (2003) 233–247 243

Fig. 6. Mean dissolved oxygen concentration (±95% confidence intervals), in mg/l, measured in input water and in outputs from ornamental
plant and reference cells at 2 and 1 day of hydraulic retention time (HRT).

reference cells (Fig. 3). The lower concentration of DO
in the planted cells indicates that oxygen was being
consumed for transformation of ammonium to nitrate.
This difference in DO is not due to COD as the removal
of COD was approximately equal in both planted and
unplanted cells.
3.4. Eh and pH
The measurements of pH showed that the influent
was slightly alkaline, with a pH around 7.7 (Table 4).
It was observed that the planted cells modified the pH
to values close to neutrality, while the unplanted cells
did not (Fig. 7). There was no significant difference
between the pH of the effluent and the influent in ref-
erence cells (P = 0.52 for HRT = 2 days, and P =
0.46 for HRT = 1 day). The difference between the
pH of the influent and the pH of the effluent from the
calla lily cells was statistically significant (P < 0.001
for both treatments). The ornamental plants had a clear
influence on the neutralization of pH in the wetland.
The redox potential (Eh) was increased in both
treatments. This observation matches the observed
elevation of DO in the wetland (Table 4). There was
a statistically significant difference between the Eh
in the influent and effluent in calla lily cells and
reference cells (P = 0.002 for HRT = 2 days, and
P < 0.001 for HRT = 1 day). There was no statisti-
cally significant difference between Eh in the effluent
from the reference and planted cells.
3.5. Nonylphenol and nonylphenol ethoxylates
Fig. 8 presents the results for the analyses of NP and
NPEOs in water from experiment 3. Data were divided
into concentrations of nonylphenol and nonylphe-
nol ethoxylates with 1–3 ethoxy units (NP(1–3)EO).
Note that, as was mentioned before, the commercial

Fig. 7. Mean pH (±95% confidence intervals), measured in input water and in outputs from ornamental plant and reference cells at 2 and
1 day of hydraulic retention time (HRT).
244 M.A. Belmont, C.D. Metcalfe / Ecological Engineering 21 (2003) 233–247

Fig. 8. Mean NP and NPEOs concentration (±95% confidence intervals), in ␮g/l, in input water and in outputs from ornamental plant and
reference cells.

mixture added to the system consisted primarily of
NPEOs with 1–3 ethoxy units. The results presented
in Fig. 8 are the average concentrations and the
95% confidence interval. Fig. 9 presents the percent-
age reduction of NP and NP(1–3)EO. The wetland
reduced appreciably the concentration of NP and
NP(1–3)EO in the water. There was a higher reduc-
tion of NP(1–3)EO than NP in both treatments. The
difference in the removal rates is partially due to the
fact that NP(1–3)EO is microbially converted to NP
in the system. This statement is supported by the
observation that there was a change in the proportion
of NP relative to NP(1–3)EO indicating conversion
of NPEOs to NP (Fig. 8). It is possible that reduc-
tion in concentrations is mainly due to adsorption of
these compounds on the substrate in the cells, since
these compounds are relatively hydrophobic, and the
shorter the ethoxy chain, the more hydrophobic the
compound.
There was high NPEO removal capacity in the
lab-scale wetland. The system reduced NP(1–3)EO
concentrations from 441.07 ± 63.71 ␮g/l in the in-
fluent to 13.00 ± 2.45 ␮g/l in the calla lily cells
(96.6 ± 0.72% reduction) and to 6.23 ± 3.22 ␮g/l
in the reference cells (98.7 ± 0.9% reduction). The
reference cells remove NP(1–3)EO slightly more effi-
ciently than the calla lily cells, the ANOVA performed
on these data showed that this difference is statisti-
cally significant (P < 0.001). There is no statistically
significant difference (P = 0.621) between the NP

Fig. 9. Mean percentage reduction of NP and NPEOs (±95% confidence intervals) in ornamental plant and reference cells in the lab-scale
wetland.
 M.A. Belmont, C.D. Metcalfe / Ecological Engineering 21 (2003) 233–247 245

removal rate in the reference cells (57.5 ± 12.7%) Table 5

and the cells planted with calla lily (54.1 ± 7.0%). Range of concentrations (␮g/l) of NP and NPEO in the lab-scale
wetland compared to primary, secondary and tertiary treated
It appears that calla lilies do not contribute to the wastewater or after passing an oxidation lagoon in Canadian mu-
removal of nonylphenol ethoxylates in this system. nicipal wastewater treatment plants (MWWTP)
Even though the percentage reduction for NP is lower
NP NP1EO NP2EO
than that for NP(1–3)EO, it is interesting to note
that NP was reduced to concentrations similar to the Primary <0.02–62.08 0.07–56.13 0.34–36.33
ones observed for NP(1–3)EO: 14.80 ± 6.04 ␮g/l for Secondary 0.12–4.79 <0.02–43.37 <0.02–32.62
Tertiary <0.02–3.20 0.30–26.4 0.25–12.45
NP in the ornamental cells (13.00 ± 2.45 ␮g/l for Lagoon 0.75–2.15 0.34–0.90 0.03–0.90
NP(1–3)EO) and 7.69 ± 2.41 ␮g/l for NP in the ref- Lab-scale influenta 25.5–41.5 241–289 103–168
erence cells (6.23 ± 3.22 ␮g/l for NP(1–3)EO). The Calla lily effluenta 11.7–28.0 5.90–18.3 4.35–6.44
lab-scale wetland did not reduce NP as well as it Reference effluenta 5.28–10.1 0.79–2.47 1.21–3.66
removed NP(1–3)EO. This was probably due to the Adapted from Bennie et al. (1998).
a 95% confidence interval.
conversion of NPEO to NP in the lab-scale wetland,
which increases the concentration of NP in the system.
A comparison between the concentrations of
nonylphenol and nonylphenol ethoxylates in this ex- least as well as a conventional primary, secondary and
periment and the values reported in the literature for tertiary MWWTP. On the other hand, the concentra-
MWWTP effluents in Canada (Bennie, 1999) shows tions of NP, NP1EO and NP2EO in the effluent of the
that the influent concentrations of NP in the lab-scale lab-scale wetland are greater than the concentrations
wetland fall into the range observed in the effluent in oxidation lagoon effluent (Table 5), showing that
of MWWTPs after primary treatment; and is greater the lab-scale wetland was less efficient at reducing
than the values observed for secondary and tertiary NP, NP1EO and NP2EO than oxidation lagoons, as
treatment plants and oxidation lagoons (Table 5). The reported by Bennie et al. (1998).
effluent concentration of NP in the lab-scale wetland Ammonium, nitrate, COD, pH and Eh were kept at
was lower than the concentrations observed in the similar levels as in the previous parts of the lab-scale
effluents of primary treatment MWWTPs (Bennie, study (Table 6).
1999), but greater than the concentrations in the ef-
fluents of secondary and tertiary treatment plants and 3.6. Limitations
oxidation lagoons (Table 5).
The influent concentrations of NP1EO and NP2EO Several of the parameters measured were highly
in the lab-scale wetland are much greater than the variable; especially in the influent. This may be due to
concentrations of NP1EO and NP2EO observed in the difficulty in keeping consistently the low flow rates
the effluents of MWWTPs (Table 5). The concentra- required for this small system. Another limitation of
tions of NP1EO and NP2EO in the effluent of the the lab-scale system is that the plants are not exposed
lab-scale wetland are smaller than the concentrations to the full range of physical and biological parameters
in the effluents of primary, secondary and tertiary in natural ecosystems (e.g. wind, rain, frost, pests,
MWWTPs (Table 5). These results showed that the wildlife, etc.). However, it was useful to reduce the
pilot-scale wetland reduced NP1EO and NP2EO at number of uncontrolled variables to focus the study on

Table 6
Ammonium, nitrate, COD, pH and Eh in the lab-scale wetland during the study of NPEO surfactant removal
Influent Effluent calla lily Effluent reference

Ammonium (mg/l) 33.6 ± 10.9 (n = 37) 13.6 ± 2.3 (n = 71) 16.9 ± 3.5 (n = 36)
Nitrate (mg/l) 3.6 ± 1.0 (n = 10) 3.4 ± 0.4 (n = 14) 18.5 ± 6.4 (n = 9)
COD (mg/l) 69.7 ± 7.7 (n = 36) 59.7 ± 4.8 (n = 59) 47.9 ± 5.4 (n = 30)
pH 7.8 ± 0.4 (n = 4) 7.6 ± 0.1 (n = 8) 7.9 ± 0.2 (n = 4)
Eh (mV) 189 ± 4.6 (n = 4) 193.8 ± 3.9 (n = 8) 185.0 ± 3.8 (n = 4)
246 M.A. Belmont, C.D. Metcalfe / Ecological Engineering 21 (2003) 233–247
some factors of the treatment process. These results
need to be verified at the field-scale system. The infor-
mation collected by lab-scale studies can be very use-
ful, but it is necessary to perform pilot-scale studies at
the location of interest, in order to have enough infor-
mation to design an appropriate full-scale treatment
wetland and minimize the chances of failure (Kadlec
and Knight, 1996).
4. Conclusions
Under proper conditions of hydraulic retention time,
calla lily can be used in subsurface flow wetlands to
reduce levels of nitrogen. The presence of these or-
namental plants was a benefit for removal of ammo-
nium through nitrification to nitrate. This suggests that
the ornamental plants promote oxygenation through
the root system, resulting in a mixture of aerobic and
anaerobic zones that favored the microbial process of
nitrification, as has been observed for wetland plants
like cattail, reed and bulrush (Reddy et al., 1989;
Kadlec and Knight, 1996). The DO was lower in the
planted cells than in the reference cells because there
was greater nitrification in the planted cells, which
consumed oxygen.
The artificial wetland regulated the pH of the influ-
ent and the plants assisted in this process. There was
no noticeable influence of ornamental plants on the
removal of COD in the wetland.
The wetland reduced the concentration of NP and
NPEOs in the surface water. The reduction of NPEOs
was higher than the reduction of NP; probably because
NP was being generated in the wetland through partial
microbial degradation of NPEOs. The proportion of
NP relative to NPEOs increased, consistent with the
conversion of NPEOs to NP in the system. It was
observed that the removal rate of the planted cells was
slightly lower than the removal rate of the reference
cells, indicating that the plants do not contribute to
removal of these surfactants in the wetland.
Acknowledgements
The authors would like to thank Richard Hughes
for his help with the analyses of NP and NPEOs. This
project was funded by a grant from the Canadian In-
ternational Development Agency to Trent University
(i.e., INSTRUCT project). Graduate stipend support
for M. Belmont was provided by the Mexican Coun-
cil of Science and Technology (Consejo Nacional de
Ciencia y Tecnologı́a, CONACyT México).
References
Ahel, M., Giger, W., Koch, M., 1986. In: Bjorseth, A., Angeletti,
G. (Eds.), Organic Micropollutants in the Aquatic Environment.
D. Reidel Publishing Co., Dordrecht, The Netherlands,
pp. 414–428.
Ahel, M., Giger, W., Koch, M., 1994a. Behavior of alkylphenol
polyethoxylate surfactants in the aquatic environment—I.
Occurrence and transformation in sewage treatment. Water Res.
28, 1131–1142.
Ahel, M., Hrsak, D., Giger, W., 1994b. Aerobic transformation
of short-chain alkylphenol polyethoxylates by mixed bacterial
cultures. Arch. Environ. Contam. Toxicol. 26, 540–548.
APHA, AWWA, WPCF, 1998. Standard Methods for the
Examination of Water and Wastewater, 20th ed. APHA,
Washington, DC.
Auman, C.W., 1996. Cultivos de flores de corte menores. In:
Larson, R.A. (Ed.), Introducción a la Floricultura. AGT Editor,
s.a. Mexico, pp. 185–186.
Bennett, E.R., Metcalfe, C.D., 2000. Distribution of degradation
products of alkylphenol ethoxylates near sewage treatment
plants in the lower Great Lakes, North America. Environ.
Toxicol. Chem. 19, 784–792.
Bennie, D.T., 1999. Review of the environmental occurrence of
alkylphenols and alkylphenol ethoxylates. Water Qual. Res. J.
Can. 34, 79–122.
Bennie, D.T., Sullivan, C.A., Lee, H.B., Maguire, R.J., 1998.
Alkylphenol polyethoxylate metabolites in Canadian sewage
treatment plant effluent streams. Water Qual. Res. J. Can. 33,
231–252.
Denny, P., 1997. Implementation of constructed wetlands in
developing countries. Water Sci. Tech. 35, 27–34.
Di Corcia, A., Samperi, R., Marcomini, A., 1994. Monitoring
aromatic surfactants and their biodegradation intermediates in
raw and treated sewages by solid phase extraction and liquid
chromatography. Environ. Sci. Technol. 28, 850–858.
Ferguson, P.L., Iden, C.R., Brownawell, B.J., 2000. Analysis of
alkylphenol ethoxylate metabolities in the aquatic environment
using liquid chromatography–electrospray mass spectrometry.
Anal. Chem. 72, 4322–4330.
Giger, W., Brunner, P.H., Schaffer, C., 1984. 4-Nonylphenol
in sewage sludge: accumulation of toxic metabolites from
nonionic surfactants. Science 225, 623–625.
Gray, M.A., Metcalfe, C.D., 1997. Induction of testes-ova in
Japanese medaka (Oryzias latipes) exposed to p-nonylphenol.
Environ. Toxicol. Chem. 16, 1082–1086.
Jobling, S., Beresford, N., Nolan, M., Rodgers-Gray, T., Brighty,
G.C., Sumpter, J.P., Tyler, C.R., 2002. Altered sexual
 M.A. Belmont, C.D. Metcalfe / Ecological Engineering 21 (2003) 233–247
maturation and gamete production in wild Roach (Rutilus
rutilus) living in rivers that receive treated sewage effluents.
Biol. Reprod. 66 (2), 272–281.
Johnson, A.C., Sumpter, J.P., 2001. Removal of endocrine-
disrupting chemicals in activated sludge treatment works.
Environ. Sci. Technol. 24 (35), 4697–4703.
Kadlec, R.H., Knight, R.L., 1996. Treatment Wetlands. CRC Press,
Lewis Publishers, Boca Raton, FL, 893 pp.
Kivaisi, A.K., 2001. The potential for constructed wetlands for
wastewater treatment and reuse in developing countries: a
review. Ecol. Eng. 16, 545–560.
Lakshman G., 1987. Ecotechnological opportunities for aquatic
plants, a survey of utilization options. In: Reddy, K.R., Smith,
W.H. (Eds.), Aquatic Plants for Water Treatment and Resource
Recovery. Magnolia Publishing Inc., Orlando, FL, pp. 49–68.
Metcalf and Eddy Inc., 1991. Wastewater Engineering. Treatment,
Disposal and Reuse, third ed. MacGraw-Hill Inc., NY, 1333
pp.
Metcalfe, C.D., Metcalfe, T.L., Kiparissis, Y., Koenig, B.G., Khan,
C., Hughes, R.J., Croley, T.R., March, R.E., Potter, T., 2001.
Estrogenic potency of chemicals detected in sewage treatment
plants effluents as determined by in vivo assays with Japanese
medaka (Orzias latipes). Environ. Toxicol. Chem. 20, 297–308.
Reddy, K.R., Patrick, W.H., Lindau, C.W., 1989. Nitrification–
denitrification at the plant root sediment interface in a flooded
organic soil. Ecol. Model. 51, 205–216.
247
Routledge, E.J., Sumpter, J.P., 1997. Structural features of
alkylphenolic chemicals associated with estrogenic activity. J.
Biol. Chem. 272, 3280–3288.
Solé, M., López-de-Alda, M.J., Castillo, M., Porte, C., Ladegaard-
Pedersen, K., Barceló, D., 2000. Estrogenicity determination
in sewage treatment plants and surface waters from the
Catalonian area (NE Spain). Environ. Sci. Technol. 34, 5076–
5083.
Stephanou, E., Giger, W., 1982. Persistent organic chemicals
in sewage effluents. 2. Quantitative determinations of nony-
lphenols and nonylphenol ethoxylates by glass capillary gas
chromatography. Environ. Sci. Technol. 16, 800–805.
Tolls, J., Kloepper-Sams, P., Sijm, D., 1994. Surfactant
bioconcentration—a critical review. Chemosphere 29, 693–
717.
Weinberger, P., Greenhalg, R., 1984. In: Garner, W.Y., Harvey,
J. (Eds.), Chemical and Biological Controls in Forestry,
ACS Symposium Series 238. American Chemical Society,
Washington, DC, pp. 351–363.
White, R., Jobling, S., Hoare, S.A., Sumpter, J.P., Parker, M.G.,
1994. Environmentally persistent alkylphenolic compounds are
estrogenic. Endocrinology 135, 175–182.
Wolverton, B.C., 1990. Aquatic plant/microbial filters for treating
septic tank effluent. In: Hammer, D.A. (Ed.), Constructed
Wetlands for Wastewater Treatment. Lewis Publishers, NY.