Science of the Total Environment 430 (2012) 119–125
Contents lists available at SciVerse ScienceDirect
Science of the Total Environment
journal homepage: www.elsevier.com/locate/scitotenv
The occurrence of steroidal estrogens in south-eastern Ontario wastewater
treatment plants
Susanna K. Atkinson ⁎, Vicki L. Marlatt, Lynda E. Kimpe, David R.S. Lean, Vance L. Trudeau, Jules M. Blais
Centre for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, ON, Canada K1N 6N5
a r t i c l e
i n f o
Article history:
Received 10 October 2011
Received in revised form 24 April 2012
Accepted 28 April 2012
Available online 25 May 2012
Keywords:
Steroidal estrogen
Biodegradation
Wastewater treatment plant
Removal efficiency
Estrogenic potency
a b s t r a c t
We measured steroidal estrogens in wastewater in Ottawa and Cornwall (Ontario, Canada) to determine removal
efficiency of these steroids during the treatment process, and whether removal varies during a seasonal cycle. Estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) were found at maximum concentrations in raw
sewage (RS), at 104, 66.9 and 5.7 ng L− 1, respectively. For the Ottawa wastewater treatment plant (WWTP),
there was sufficient data to show that E1 concentrations in RS correlated with both ambient air temperature
and mean daily flow of the WWTP (R2 =0.792, p =0.003 and R2 =0.757, p =0.005). E1 removal was correlated
with the percent difference in cBOD from RS to FE (final effluent) (R2 =0.435, p = 0.075). However estrogenic potency, as determined by a sensitive in vitro reporter gene assay, did not decrease during the water treatment process, suggesting that many estrogenic chemicals are conserved in FE. E1 and EE2 were found in river water, both
upstream and downstream of the WWTPs, and at much lower concentrations than in FE. Our study demonstrates
the persistence of steroidal estrogens and estrogenic potency in Ontario WWTP effluents and surface waters, and
has uncovered temporal patterns of release that may be used to help predict risks to aquatic organisms in these
environments.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
The excretion of steroidal hormones from human populations is a
major contributor to the presence of endocrine-disrupting chemicals
(EDCs) in the aquatic environment. 17β-estradiol (E2), the major estrogen in vertebrates, estrone (E1), the precursor as well as a breakdown
product of E2, and 17α-ethinylestradiol (EE2), the synthetic estrogen
used in the birth control pill, are released in conjugated forms in urine
(Ternes et al., 1999b). Their principal entry into the environment is via
wastewater treatment plant (WWTP) effluents. Chemical fractionation
and in vitro biological screening with yeast have identified E1, E2 and
EE2 as the greatest sources of estrogenic activity in domestic sewage effluents (Desbrow et al., 1998).
Moreover, significant progress has been made in quantifying the presence of steroidal estrogens in waste and surface waters, which have been
measured in the ng L− 1 to μg L− 1 range (Williams et al., 2003; Ternes et
al., 1999a; Carbella et al., 2004; Barel-Cohen et al., 2006; Fernandez et al.,
2007; Lishman et al., 2006). The next challenge is to quantify the
⁎ Corresponding author at: Pest Management Regulatory Agency, Health Canada,
2720 Riverside Drive, Ottawa, ON, Canada K1S 0M2. Tel.: + 1 613 736 3898; fax: + 1
613 736 3540.
E-mail addresses: susannakatkinson@gmail.com (S.K. Atkinson),
vicki.marlatt@ufv.ca (V.L. Marlatt), lkimpe@uottawa.ca (L.E. Kimpe),
drslean@gmail.com (D.R.S. Lean), trudeauv@uottawa.ca (V.L. Trudeau),
jules.blais@uottawa.ca (J.M. Blais).
0048-9697/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.scitotenv.2012.04.069
persistence and patterns of degradation of estrogens in these environments, enabling the estimation and reduction of risks to aquatic
organisms.
The degradation of steroidal estrogens is thought to be microbially
mediated, with deconjugation of sulfates and glucuronides and oxidation of E2 and EE2 into E1 as the principal transformations (Carbella
et al., 2004). Microbial activity in a WWTP may therefore be a proxy
to predict elimination of steroids from raw sewage to final effluent
(Johnson and Sumpter, 2001). This activity is often measured as the carbonaceous biochemical oxygen demand (cBOD), which varies greatly
with the type of treatment (activated sludge, biological trickling filter, ultraviolet treatment) (Kirk et al., 2001; Carbella et al., 2004; Schlüsener
and Bester, 2008). Increased hydraulic and sludge retention times
(HRTs and SRTs), which allow time for the biological reactions to occur,
and increased temperatures, which promote microbial degradation,
can increase steroid elimination rates (Lee and Liu, 2002; Lishman et
al., 2006; Shiwei et al., 2008; Ternes et al., 1999a). Despite these degradation reactions in WWTPs, steroidal estrogens continue to be detected in
the natural aquatic environment. Barel-Cohen et al. (2006) determined
that although natural and synthetic estrogen concentrations were reduced by half within 25 km from a sewage effluent source, they could
still be detected up to 100 km along the river. The most potent but often
least abundant steroidal estrogen EE2 has even been found in German
drinking water at 0.5 ng L− 1 (Aherne and Briggs, 1989).
The purpose of this study was (1) to quantify steroidal estrogens
in the waste and receiving surface waters surrounding two southeastern
Ontario WWTPs, in Ottawa and Cornwall, Ontario Canada; and (2) to
120
S.K. Atkinson et al. / Science of the Total Environment 430 (2012) 119–125
Table 1
Latitude, longitude and altitude of sampling locations.
Sampling location
WWTP
Ottawa
River
Cornwall
Ottawa
St. Lawrence
Raw sewage
Final effluent
N/A
Upstream
Downstream
Site 1
Site 2
Latitude
(N)
Longitude
(W)
Altitude
(masl)
45°
45°
45°
45°
45°
45°
45°
75°
75°
74°
75°
75°
74°
74°
54
52
65
40
42
47
46
27′
27′
01′
27′
28′
01′
01′
5.69″
50.64″
46.21″
55.97″
45.35″
06.57″
29.82″
35′
35′
40′
35′
33′
41′
40′
29.29″
33.32″
42.39″
46.57″
37.52″
45.74″
56.87″
Coordinates and altitudes obtained from Google Earth (2007).
masl = meters above sea level.
determine whether the efficiency of steroid removal by WWTPs relates
to treatment type, HRT and temperature. We also incorporated precipitation in our model, as it may affect HRTs, and, like temperature, will vary
seasonally.
We predicted that E1, E2 and EE2 loadings would be greater to the
Ottawa WWTP than the Cornwall WWTP, because of the larger population being served. Furthermore, we hypothesized that steroidal estrogen
removal would be more efficient in the Ottawa WWTP than the Cornwall
WWTP because Ottawa uses secondary treatment whereas Cornwall
uses only primary treatment. Finally, we predicted that E1 would be
the least persistent of the three estrogens, and that EE2 would be the
most persistent.
2. Methods
2.1. Study sites
The City of Ottawa WWTP is a secondary treatment facility with
year round activated sludge and phosphorus removal, and seasonal
chlorine disinfection occurring from May 16th to November 15th
(Sierra Legal, 2004). Total daily flow is 4.22 × 10 8 L (Ontario Centre for
Municipal Best Practices, 2006). The City of Cornwall WWTP is a primary treatment facility with a total daily flow of 1.63 × 107 L (United States
Environmental Protection Agency, 2008; Department of Infrastructure
& Municipal Works, City of Cornwall, personal communications,
2008). The locations of the study sites are given in Table 1.
and the Laboratory Services of the Environmental Programs and Technical Support (personal communications, 2006; Appendix A). Details
on the sampling and analysis of the hydrology and water chemistry
data at the Ottawa WWTP can be found in its Annual Operations Report (City of Ottawa, 2005).
2.3. Ottawa meteorological data
The daily mean ambient air temperature and total precipitation for
the City of Ottawa were collected for the five days prior to each Ottawa
wastewater sampling date (National Climate Data and Information
Archive, 2011). For each sampling event, a mean temperature was calculated and a sum for the total daily precipitation was tabulated (Appendix B). The five days chosen to calculate the mean temperature
and total precipitation for each sampling event was an estimate of the
average time it takes for City of Ottawa RS to arrive at the WWTP.
2.4. Prediction of estrogen concentrations entering WWTPs
Estimates of the population being served by the Ottawa and Cornwall
WWTPs were obtained from 2006 Community Profiles (2011) (Appendices C and D). While 96.8% of the City of Ottawa population is served
by the Ottawa wastewater treatment plant (Ontario Centre for
Municipal Best Practices, 2006), the population being served by the
Cornwall wastewater treatment plant was assumed to be the total population of the City of Cornwall.
Population data were separated into groups of males and females,
with the latter divided into pre-menstruating, menstruating, menopausal and pregnant women (Tables 2 and 3). There was a further subdivision of women taking the contraceptive pill, and women on
hormone replacement therapy. Menstruating women were defined as
females between the ages of 15 and 59 years, and the proportions of
the female population that are pregnant (1/75), use the contraceptive
pill (13%) and are on hormone replacement therapy (3%) were defined
as reported by Johnson et al. (2000). As well, it was assumed that premenstruating females release the same amounts of E1 and E2 as males.
This information, along with the steroidal estrogen excretion rates
described by Johnson et al. (2000) and Christiansen et al. (2002), was
used to calculate the total daily loadings of E1, E2 and EE2 in raw sewages
in the Ottawa and Cornwall WWTPs. The daily loadings of estrogens
were then divided by the average daily flow in the WWTPs to predict
concentrations of E1, E2 and EE2 entering the plants (Tables 2 and 3).
2.2. Hydrology and water chemistry at the Ottawa WWTP
2.5. Sampling, extraction and analysis
The daily WWTP flow (Q), hydraulic retention time (HRT), FE
temperature (FET), and RS and FE carbonaceous biochemical oxygen
demand (cBOD) for the wastewater sampling dates were obtained
from monitoring records provided by the Ottawa WWTP technicians
2.5.1. Sampling and isotope dilution
Triplicate grab wastewater composite samples were taken between
June 2005 and August 2006 in 1-L amber glass bottles. An isotope
Table 2
Predicted concentrations of estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) in Ottawa raw sewage.
Study
Group
Population
E1 excreted daily (μg)
E2 excreted daily (μg)
EE2 excreted daily (μg)
Johnson et al. (2000)
Males
Pre-menstruating females
Menstruating females
Menopausal women
Pregnant women
Women on pill
Women on hormone therapy
Total
Predicted concentration (ng L− 1)
380,418
67,880
257,050
75,362
5409
34,120
7874
1.48 × 106
2.65 × 105
2.06 × 106
3.01 × 105
1.40 × 106
n/a
n/a
5.51 × 106
13.1
6.09 × 105
1.09 × 105
1.23 × 106
1.73 × 105
3.25 × 106
n/a
n/a
5.37 × 106
12.7
n/a
n/a
n/a
n/a
n/a
3.10 × 105
5.63 × 104
3.67 × 105
0.9
Christiansen et al. (2002)
Total
Predicted concentration (ng L− 1)
786,130
1.01 × 107
23.9
5.25 × 106
12.4
4.70 × 105
1.1
Data based on Johnson et al. (2000), Christiansen et al. (2002), 2006 Community Profiles (2011) and Ontario Centre for Municipal Best Practices (2006); an average daily flow of
4.22 × 108 L was used.
n/a = not applicable.
S.K. Atkinson et al. / Science of the Total Environment 430 (2012) 119–125
121
Table 3
Predicted concentrations of estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) in Cornwall raw sewage.
Study
Group
Population
E1 excreted daily (μg)
E2 excreted daily (μg)
EE2 excreted daily (μg)
Johnson et al. (2000)
Males
Pre-menstruating females
Menstruating females
Menopausal women
Pregnant women
Women on pill
Women on hormone therapy
Total
Predicted concentration (ng L− 1)
21,795
3810
13,683
6345
322
1821
420
8.50 × 104
1.49 × 104
1.09 × 105
2.54 × 104
8.35 × 104
n/a
n/a
3.18 × 105
7.3
3.49 × 104
6.10 × 103
6.57 × 104
1.46 × 104
1.93 × 105
n/a
n/a
3.15 × 105
7.2
n/a
n/a
n/a
n/a
n/a
1.66 × 104
3.00 × 103
1.96 × 104
0.4
Christiansen et al. (2002)
Total
Predicted concentration (ng L− 1)
45,965
5.89 × 105
13.5
3.07 × 105
7.0
2.75 × 104
0.6
Data based on Johnson et al. (2000), Christiansen et al. (2002), 2006 Community Profiles (2011) and personal communications with the Department of Infrastructure & Municipal
Works, City of Cornwall (2008); an average daily flow of 1.63 × 107 L was used.
n/a = not applicable.
dilution was performed immediately after sampling by adding 80 μL of
a 100 μg mL− 1 internal standard of 13C-labeled E1, E2 and EE2 in acetonitrile (E1: 3, 4-13C, 90% purity; E2: 3, 4-13C, 99% purity; EE2: 20, 21-13C,
99% purity; Cambridge Isotope Laboratories, Andover, MA). Adding the
13
C-labeled standards prior to filtration means the analysis provides the
total amount in all phases, providing the basic assumption, that the isotopes added are distributed in the same way as the native compounds,
is fulfilled (de Hoffman and Stroobant, 2002).
2.5.2. Filtration and solid phase extraction
Filtration and extraction of samples were based on Lee and Peart's
method (1998). Samples were filtered using glass fiber filters
(Whatman GF/F 47 mm) and celite (Aldrich Grade 545), when needed,
to prevent the filter from plugging. Samples were vacuum-loaded onto
1 g ENVI-18 tube solid phase extraction (SPE) tubes (Sigma-Aldrich,
Oakville, ON) pre-conditioned with 5 mL acetone, 5 mL methanol and
10 mL high-performance liquid chromatography (HPLC) water (HPLC,
GC pesticide grade; Burdick & Jackson, Muskegon, MI) using Visiprep
large-volume sampler 1/8 in. Teflon tubes and an ENVI-Disk Holder
manifold for 6 × 1 L samples (both from Sigma-Aldrich, Oakville, ON).
The SPE tubes were eluted with 5 mL acetone and 5 mL methanol. An
average flow rate of 10 mL/min was maintained for the pre-conditioned
elution, the water sample, and the elution. Extracts were evaporated to
dryness under a stream of nitrogen gas using a mini-evaporator.
2.5.3. Derivatization and clean-up
Residues were redissolved in 2 mL HPLC water and a derivatization
method was done by adding 10 μL 10% pentafluorobenzoyl chloride
(2, 3, 4, 5, 6 — PFBCl) (Sigma-Aldrich, Oakville, ON) in toluene (Omnisolve, 99.98% purity; EMD Chemicals, Gibbstown, NJ) and 50 μL 2 M potassium hydroxide (KOH) buffer in water (Sigma-Aldrich, Oakville, ON).
The mixture was then extracted twice with 2.5 mL hexane (HPLC, GC
pesticide grade; Burdrick & Jackson, Muskegon, MI) and reduced to
0.5 mL with the mini-evaporator. Subsequently, the organic phase was
passed through a glass pipette column plugged with glass wool (presonicated in dichloromethane, DCM) and filled with anhydrous sodium
sulfate (ACS Grade, EMD granular) to remove impurities and any traces
of water from the HPLC water and KOH buffer in water used in the derivatization reaction. The sample was dried with the mini-evaporator
and resuspended in 200 μL isooctane (2,2,4-trimethylpentane, optima
grade, 99% purity; Fisher Scientific, Fair Lawn, NJ).
2.5.4. Separation and detection by GC/MS
The pentafluorobenzoyl-derivatives of E1, E2 and EE2 were separated
and detected by gas chromatography–mass spectrometry (GC−MS).
Samples of 4 μL were injected in splitless mode into a gas chromatograph
with a Zebron ZB-5 ms column (25 m×250.00 μm×0.25 μm), a 10 cm
guard, and a helium carrier gas at 65 cm s− 1 (6890 series; Agilent Technologies, Palo Alto, CA). The injector was set at 250 °C and the oven
temperatures were programmed as follows: 80 °C min− 1 for 1 min,
24 °C min− 1 until 200 °C and held for 0.5 min, 73 °C min− 1 until
245 °C and held for 5 min, and then 1 °C min− 1 to 260 °C. The mass
spec transfer line temperature was set at 300 °C. A Hewlett Packard
5973 quadrupole mass spectrometer and chemical ion source (Palo
Alto, CA), with the quad temperature set at 100 °C, the source at
200 °C, and the methane reaction gas at 40% (26 cm s− 1), were used
for quantification of estrogens in negative chemical ionization (NCI)
mode. The following quantification ions were used in selected ion monitoring (SIM) mode: m/z 464 (C-12 E1), m/z 466 (C-13 E1, C-12 E2), m/z
468 (C-13 E2), m/z 490 (C-12 EE2) and m/z 492 (C-13 EE2). Since the
13
C-labeled standards were added to each sample prior to workup, any
problems with recovery that may have occurred during workup and
analysis should be compensated by isotopic dilution. Supplementary
identifier masses and characteristic fragments were used in selected
samples for analyte confirmation of E1 and EE2 following methods in
Kuch and Ballschmiter (2001). Chromatographic data were collected
and analyzed using Agilent MSD Chemstation data analysis software
(Palo Alto, CA).
2.5.5. Detection limits
Instrument detection limits (IDLs) and method detection limits
(MDLs) were calculated using the methods outlined by the United
States Environmental Protection Agency (1993). IDLs were obtained
from seven injections of one water sample extract (prepared from E1,
E2 and EE2 standards of 40, 80 and 5 ng L − 1, respectively, in 1 L double
distilled water) where the standard deviation for the mean concentration was multiplied by three. IDLs for E1, E2 and EE2 were 1.2, 14.2
and 0.4 ng L − 1, respectively. MDLs were obtained by the injection of
seven extracts from seven water samples (prepared from E1, E2 and
EE2 standards of 5, 5 and 1 ng L − 1, respectively, in 1 L double distilled
water) extracted identically, where the standard deviation for the
mean concentration was multiplied by the one-tailed t statistic at a
95% confidence level. MDLs for E1, E2 and EE2 were 1.8, 24.3 and
0.5 ng L− 1, respectively. The detection limits for the extraction of E2
from water was elevated due to difficulty in separating E1 and E2.
In addition, the linear responses of steroidal estrogens were confirmed from sample extracts of 0, 12.5, 25, 37.5 ng L− 1 of E1 and E2
standards and from sample extracts of 0, 1, 2, 3 and 4 ng L − 1 of EE2
standards. Plotting the responses versus the concentration gave R 2
values of 0.96, 0.97 and 0.99 for E1, E2 and EE2, respectively. Blank
samples of double distilled water were spiked with the 13C standards
122
S.K. Atkinson et al. / Science of the Total Environment 430 (2012) 119–125
β-glucuronidase, as well as the oxidation of E2 to E1 (D'Ascenzo et al.,
2003; Ternes et al., 1999b; Lee and Liu, 2002).
Other estrogens (E2 and EE2) were less frequently detected in this
study, as has been seen elsewhere in the literature (Carbella et al., 2004;
Fernandez et al., 2007). E2 was detected consistently in RS and FE samples from both WWTPs, but concentrations were much lower than for
E1, and only quantifiable in three samples. E2 was found at a maximum
concentration of 66.9 ng L − 1 (SE ± 15.7 ng L − 1) in Ottawa RS on
September 14, 2005 (Table 4). EE2 was detected in every Cornwall
WWTP sample, at a maximum concentration of 9.8 ng L− 1 (SE±
8.4 ng L− 1) in FE from June 29, 2005 (Table 4). In Ottawa, EE2 was only
detected in two samples, one RS and one FE, from two different sampling
dates. The sporadic occurrences of EE2 are similar to those reported by
Williams et al. (2003), and may be because it is not released at such an
elevated and constant rate from a population, compared to the other
estrogens.
Aside from their degradation, estrogens may not be detected in
wastewater because they have sorbed to sewage sludge, which may
be difficult to gather in a composite sample. EE2 in particular, which
has the highest Kow (1.41 × 104 compared to 2.69 × 103 for E1 and
8.71× 103 for E2), shows this tendency (Suidan et al., 2005; Lai et al.,
2002). Difficulties in analyzing sludge-bound estrogens may prevent a
complete mass-balance analysis of E1, E2 and EE2 around each treatment unit in a plant. That concentrations in a sewage sludge are not
well studied is a concern, because sludge is frequently applied as fertilizer
to agricultural lands.
(Cambridge Isotope Laboratories, Andover, MA) and taken through the
extraction process alongside each series of sample analysis.
2.6. In vitro estrogenicity assay
Four Ottawa wastewater samples (2 RS and 2 FE) collected on October
11, 2005 were analyzed for estrogen concentrations, as described in
Section 2.5. A second set of wastewater samples (again, 2 RS and 2 FE),
along with two positive E1 controls (500 ng L− 1), were only filtered
and passed through an SPE tube, as described in Section 2.5.2, omitting
the isotope dilution step. The dried residues of the four samples and
two controls were resuspended in 100 μL of dimethyl sulfoxide
(DMSO). Each extract in DMSO was assessed in a fish-specific, estrogen
receptor-dependent reporter gene assay, as previously reported
(Ackermann et al., 2002) and validated in our laboratory (Atkinson et
al., 2011). Briefly, in this assay the rainbow trout (Oncorhynchus mykiss)
gonad cell line RTG-2 is transiently transfected with an expression vector
containing the rainbow trout estrogen receptor alpha complimentary
DNA, an estrogen-dependent firefly luciferase reporter plasmid (pERETK-Luc) and a renilla luciferase control vector (PRL-TK). The cells are
subsequently exposed to cell culture test medium (phenol-red free) containing the DMSO solvent control or test substance dissolved in DMSO in
triplicate wells; the DMSO concentration in the cell culture test medium
per well for all treatments was 0.05%. The mean fold induction above the
DMSO control of the positive E1 control, and the samples of RS and FE,
are reported.
3.2. Comparison of predicted concentrations versus measured concentrations
3. Results and discussion
The E1 concentrations measured in raw sewage are higher than
what would be predicted with a model using the excretion values
reported by Johnson et al. (2000) and Christiansen et al. (2002) by
about a factor of 2 (Tables 2 and 3). Similarly, where the E2 concentrations are quantifiable, these are also higher than what would be
predicted by the model. Discrepancies between the predicted concentrations and the measured concentrations may be due to the different
sex ratios and age distribution of the Danish and Ottawa populations,
along with the difficulties in estimating the number of pregnancies
and number of people taking the oral contraceptive pill or undergoing
hormonal therapy, and deviations from the average daily flow
(Johnson et al., 2000). The higher daily loadings of E1 and E2 in Ottawa
wastewater compared to Cornwall are linked to the larger human population being served by the Ottawa sewage treatment plant (approximately 17 times higher than Cornwall). This is also in keeping with
3.1. Concentrations of steroidal estrogens in wastewater
The concentrations of steroidal estrogens measured in samples of
Ottawa and Cornwall RS and FE (Table 4) compare well to the values
obtained in other Canadian studies (Cicek et al., 2007; Fernandez et
al., 2007; Servos et al., 2005).
E1 was found consistently in all the RS and FE samples collected,
from both the Ottawa and Cornwall WWTPs. E1 was measured at a maximum concentration of 370 ng L− 1 (SE±26 ng L− 1) on August 16, 2006,
and at a minimum concentration of 11.2 ng L− 1 (SE± 0.9 ng L− 1) on
November 30, 2005, both in Ottawa FE samples (Table 4). Generally,
the E1 concentration increased during the treatment process. This is likely due to both the decoupling of estrogen sulfates and estrogen glucuronide conjugates by the fecal coliform bacteria (Escherichia coli) enzyme
Table 4
Concentrations of estrone, 17β-estradiol, 17α-ethinylestradiol in Ottawa and Cornwall wastewater samples; means (standard error) of raw sewage (RS) and final effluent (FE) concentrations, and percent difference (% Δ) from raw sewage to final effluent are listed.
Site
Date
Estrone
Mean (ng L
Ottawa
Cornwall
June 9/05
July 20/05
Aug. 4/05
Sept. 14/05
Oct. 11/05a
Nov. 30/05
Jan. 19/06
May 10/06
June 6/06
Aug. 16/06
June 29/05
Aug. 11/05
17β-estradiol
−1
%Δ
)
RS
FE
47.3 (4.1)
50.4 (1.5)
46.7 (1.4)
44.9 (9.3)
46.0 (3.7)
35.0 (1.7)
35.2 (0.9)
48.6 (3.0)
not sampled
104 (5.0)
13.1 (1.5)
29.3 (0.1)
86.8
20.6
205
158
113
11.2
44.9
11.3
15.7
370
16.0
22.9
(7.3)
(0.4)
(2.0)
(2.0)
(70)
(0.9)
(1.3)
(0.4)
(2.1)
(26)
(0.2)
(0.2)
− 83.5
59.1b
− 340b
− 252b
− 148
68.2b
− 27.6b
76.8b
n/a
− 257b
− 22.5
21.9b
Mean (ng L
17α-ethinylestradiol
−1
%Δ
)
RS
FE
24.7 (0.1)
b MDL
b MDL
66.9 (15.7)
b MDL
b MDL
nd
nd
not sampled
nd
b MDL
b MDL
b MDL
nd
nd
b MDL
26.7
nd
b MDL
nd
b MDL
nd
b MDL
b MDL
MDL = method detection limit; n/a = not applicable; nd = not detected.
a
Samples analyzed for inductions of reporter gene activity above a DMSO solvent control.
b
Significant difference between raw sewage and final effluent concentration.
n/a
100
100
n/a
n/a
100
n/a
n/a
n/a
n/a
n/a
n/a
Mean (ng L− 1)
%Δ
RS
FE
nd
nd
nd
nd
nd
nd
1.3 (1.3)
nd
not sampled
nd
5.7 (4.1)
0.5 (0.1)
nd
nd
nd
0.6 (0.4)
nd
nd
nd
nd
nd
nd
9.8 (8.4)
1.0 (0.1)
n/a
n/a
n/a
n/a
n/a
n/a
100
n/a
n/a
n/a
− 73.1
− 119
S.K. Atkinson et al. / Science of the Total Environment 430 (2012) 119–125
3.3. Effect of meteorological variables and WWTP parameters on steroidal
estrogen concentrations
Regression analyses were used to examine the relationships between meteorological variables/WWTP parameters and steroidal estrogen concentrations for E1, because it is the most consistently measured
steroidal estrogen and it provides the most complete data set from
which to examine patterns over time and space. Studentized residuals
were used to justify leaving the August 2006 Ottawa E1 concentrations
out of the regression analyses; concentrations were over two times
greater than for all other values.
A significant positive relationship was found between the concentrations of E1 in RS and the mean ambient air temperature (R2 = 0.792,
p = 0.003; Fig. 1); this is perhaps due to increased microbial activity at
higher temperatures. Although breakdown of E1 may occur, at this
stage of wastewater's pathway into the environment, the oxidation of
conjugates and the conversion of E2 into E1 are greater than the breakdown of E1. Shiwei et al. (2008) reported higher concentrations of E1 in
Chinese raw sewage in summer months, also attributing this to a more
rapid degradation of E2 under higher temperatures. A significant inverse
relationship was also found between E1 in RS and mean daily flow
(R2 = 0.757, p = 0.005; Fig. 2), which is likely a dilution effect.
No significant relationships were found between E1 in FE and any
meteorological variable or WWTP parameter.
55
Concentration (ng/L)
Desforges et al. (2010), who showed that estrogenic potency, as measured by plasma vitellogenin from male teleost fish in rivers around
the world, was correlated with upstream human population size.
123
50
45
R2 = 0.757
p = 0.005
40
35
30
5.8
6
6.2
6.4
6.6
6.8
LN [Q] (ML)]
Fig. 2. Concentration of estrone in Ottawa raw sewage versus mean daily flow.
The percent removal in cBOD from RS to FE explained some of the
variations observed in percent removal of E1 from RS to FE (R 2 = 0.435,
p= 0.075, Fig. 3). Advancements in upgrading sewage treatment processes are usually designed to limit biological oxygen demand (BOD),
total suspended solids (TSS) and total phosphorus in effluent, and
sometimes ammonia nitrogen, E. coli and total residual chlorine; the removal of natural hormones is generally not a criterion (Sierra Legal,
2006). Here, the use of secondary treatment for removing organic material may also promote reactions that produce E1 (namely deconjugation
of estrogen sulfates and glucorinides and oxidation of E2), and release of
E1 in FE, a trend seen in other secondary WWTPs (Carbella et al., 2004;
Schlüsener and Bester, 2008).
3.4. Removal of steroidal estrogens through the WWTP
3.5. Estrogenicity of wastewater
In the Ottawa WWTP, there is a reduction of both E1 and E2 concentrations from raw sewage to final effluent. For 7 of the 9 Ottawa sampling dates for which percent E1 removal was calculated, a two-tailed
t-test showed a significant difference (p≤ 0.05) between RS and FE. In
Ottawa, E1 concentrations increased in FE up to 340%, compared to
Cornwall FE, where the increase was only 22%. As discussed, the increase in E1, also seen in studies by Fernandez et al. (2007) and
Schlüsener and Bester (2008), is likely from microbial deconjugation
of steroidal esters and the conversion of E2 into E1, which is more efficient in the secondary plant than in the primary plant. On several dates,
removal of E2 to a non-detectable level is likely due to oxidation of E2
into E1 and its subsequent breakdown products, (Table 4). However,
secondary treatment does not seem to remove E2 more efficiently
than the primary treatment; in both plants, E2 can still be detected in
the final effluent. Due to the sporadic occurrences of EE2 found in
wastewater, no conclusion can be made about its removal in secondary
versus primary treatments.
The E1 concentration in Ottawa wastewater sampled on October
11, 2005, increased from RS (E1 = 46.0 ng L − 1 ± 3.7 ng L − 1) to FE
(113 ng L − 1 ± 70 ng L − 1, E2 b MDL, EE2 = nd), but this increase was
not significant (Table 4). Estradiol and EE2 concentrations did not
change (RS and FE: E2b MDL, EE2 = nd). Both the RS and FE samples
caused significant elevation (one-way ANOVA, Tukey's post hoc test;
p b 0.05) of estrogen-induced luciferase activity in the reporter gene
assay (4.2± 0.3 and 3.5 ± 0.4 fold, respectively; mean± standard error
(SEM)), and the magnitude of these responses was lower but not significantly different from the 500 ng L− 1 E1 positive control (8.7 ± 1.4 fold;
mean± SEM; Fig. 4; p > 0.05). In addition, the estrogenicity of the RS
compared to the FE samples was not significantly different in the reporter gene assay (Fig. 4; p > 0.05), which coincides with the absence
of significantly higher steroidal estrogens measured during the chemical analyses of the FE samples compared to the RS samples. Collectively,
these results demonstrate that sewage treatment was not very effective
200
100
55
% Difference
Concentration (ng/L)
60
50
45
40
35
30
-10
0
-100
-200
R2 = 0.435
p = 0.075
-300
R2 = 0.792
p = 0.003
-400
-5
0
5
10
15
20
25
30
Temperature (oC)
Fig. 1. Concentration of estrone in Ottawa raw sewage versus mean environmental
temperature five days prior to sampling.
90
92
94
96
98
100
% Difference cBOD
Fig. 3. Percent difference of estrone versus percent difference of carbonaceous biological
oxygen demand in Ottawa wastewater.
124
S.K. Atkinson et al. / Science of the Total Environment 430 (2012) 119–125
Fold induction above
DMSO control
12
10
8
6
4
2
0
E1 control
Raw Sewage
Final Effluent
Fig. 4. Estrogen-dependent luciferase induction by positive estrone control (500 ng L− 1),
and raw sewage and final effluent samples collected from the Ottawa WWTP in a rainbow
trout estrogen receptor reporter gene assay. Mean fold induction (± SE) above the DMSO
control of two independent experiments are presented. An ANOVA shows no significant
difference between the E1 control, the raw sewage and the final effluent samples.
in removing the estrogen bioactivity caused by compounds binding to
the trout ERα in the in vitro reporter gene assay.
Although a full dose response curve with E1 was not included in the
present study, the relative potency of E1 relative to E2 ranges from 0.1
to 0.7 in various in vitro and in vivo bioassays (Cespedes et al., 2004;
Van den Belt et al., 2004; Salste et al., 2007; Thorpe et al., 2006). These
relative potency values reported in the literature are similar to the relative potency value estimated when comparing the single 500 ng L− 1 E1
dose tested to the E2 standard curve obtained in the present experiments.
Without a complete chemical analysis of the wastewater samples tested including non-hormone based estrogenic compounds
(i.e. alkylphenols, phthalates, and pesticides), we cannot completely account for all of the compounds that are contributing to the estrogenic or
anti-estrogenic activity of the RS and FE samples. Our results suggest
that the breakdown products in a WWTP may include E1, and most likely,
additional uncharacterized compounds. This may include lumiestrone, a
little known photochemical derivative of E1 that we have recently discovered to be biologically active in vivo and in vitro (Trudeau et al.,
2011). It is possible that the levels of E1 present and additional
uncharacterized compounds in these Ottawa wastewater samples have
the potential to induce an estrogenic response in exposed aquatic wildlife. Future investigations aimed at a more complete chemical analysis
of wastewater constituents and continued monitoring of the Ottawa
WWTP for endocrine disrupting effects are warranted.
3.6. Concentrations of steroidal estrogens in rivers
E1 and EE2 were detected in both rivers at maximum concentrations
of 107 ng L− 1 (SE±35 ng L− 1) in the St. Lawrence River, and 2.2 ng L− 1
(SE ± 1.0 ng L − 1) in the Ottawa River, respectively (Table 5). Whereas
the EE2 value is within the range of measurements found elsewhere,
the E1 value is higher than measurements previously reported in surface waters (Labadie and Budzinski, 2005; Williams et al., 2003). E2
was not detected in either of the rivers.
Because sampling of both WWTP and rivers on the same day was
not often possible, river sample results are compared to final effluent
samples taken one month before, and one month after river sampling
dates. The detection of 20.6 ng L − 1 E1 in Ottawa FE on July 20, 2005,
and its absence from the downstream sample, suggest that degradation, sorption and dilution of estrogens occur in rivers. These reactions are further studied by Jürgens et al. (2002) and Barel-Cohen et
al. (2006). The high E1 concentration (107 ng L − 1 SE ± 35 ng L − 1)
measured in the St. Lawrence upstream of the Cornwall wastewater
treatment plant, is at least four times higher than any measured or
predicted concentration of E1 in Cornwall wastewater (Tables 3, 4
and 5). As well, EE2 is found upstream of both wastewater treatment
plants, even though it is never detected in raw sewage. These values
suggest that a source of estrogens exists upstream from the wastewater treatment plants, which may be from storm sewer overflow or agricultural sources. In addition to release from domestic wastewater,
E1, E2 and EE2 can also enter watersheds through runoff from land,
where sources include cattle or pig excrement and fertilizers. This
type of transport and degradation has not been well investigated in
the field (Gagné et al., 2001; Johnson and Sumpter, 2001; Khanal et
al., 2006; Johnson et al., 2006).
The river water sampling results also demonstrate the resilience of
estrogenic compounds in the natural environment, particularly
ethinylestradiol. In Ottawa and Cornwall, E1 and EE2 were detected
downstream of the wastewater treatment plant; whereas E1 was measured at much lower concentrations in downstream samples than in
final effluents, the concentrations of EE2 did not decrease. Some have hypothesized that estrogenic compounds may originate from the Ottawa
River after observing significantly higher VTG levels in mussels and
fish in Rivière Des Prairies, to which the Ottawa River drains
(Aravindakshan et al., 2004), and the present study supports this hypothesis. Whether these compounds originate from Ottawa effluent or
via runoff, these compounds exhibit environmental persistence.
4. Conclusion
We found patterns of occurrence, removal and persistence of E1,
E2 and EE2 in Ottawa and Cornwall WWTPs and surface waters. Secondary treatment does not seem to improve removal efficiency of E2 and
EE2, in comparison to primary treatment, and in addition, it also produces higher concentrations of E1 in final effluent. The production of
E1 appears to be microbially mediated because of the relationship between E1 production and cBOD removal, and may be more efficient at
higher temperatures, resulting in a seasonal pattern of estrogen release.
The sampling results suggest that in addition to wastewater effluent,
other sources of estrogens to the Ottawa River and St. Lawrence River
Table 5
Concentrations of estrone, 17β-estradiol, 17α-ethinylestradiol in Ottawa and Cornwall final effluent (FE) samples and in Ottawa and St. Lawrence River upstream and downstream
samples; means (standard error) of concentrations are listed.
Site
Date
Estrone
Mean (ng L
17β-estradiol
−1
FE
Ottawa
Cornwall
June 9/05
June 22/05
July 20/05
Aug. 4/05
June 15/05
June 29/05
86.8
–
20.6
86.8
–
16.0
(7.3)
(0.4)
(7.3)
(0.2)
MDL = method detection limit; nd = not detected.
“–” indicates that for this date, no sample was taken.
)
Mean (ng L
−1
17α-ethinylestradiol
Mean (ng L− 1)
)
US
DS
FE
US
DS
FE
US
DS
–
nd
nd
–
107 (35)
–
–
bMDL
nd
–
nd
–
b MDL
–
nd
b MDL
–
b MDL
–
nd
nd
–
nd
–
–
nd
nd
–
nd
–
nd
–
nd
nd
–
9.8 (8.4)
–
0.8 (0.3)
bMDL
–
0.4 (0.2)
–
–
2.2 (1.0)
b MDL
–
0.7 (0.1)
–
S.K. Atkinson et al. / Science of the Total Environment 430 (2012) 119–125
exist and need to be better described (e.g., farming activity, storm
sewers, coastal currents).
Continued monitoring of these steroidal estrogens is necessary in
order to improve wastewater treatment processes, and to ensure the
safety of drinking water. It is important to examine low potency steroidal estrogens (i.e. estrone) because they are commonly detected in surface waters worldwide, and can significantly contribute to the overall
estrogenic activity of a sample. Monitoring should also be expanded to
include conjugates, sludge-bound estrogens, and other estrogen agonists and antagonists. Given the persistence of estrogenicity in effluent
and rivers, we recommend surveillance studies for indicators of
estrogenization of aquatic wildlife populations within these waters.
Acknowledgments
This research was funded by grants from the National Science and
Engineering Research Council of Canada to JMB, DRSL and VLT. Thanks
are given to Gary Robidoux (City of Ottawa), Serena Maharaj (University
of Ottawa), Adrienne Fowlie and Jeff Ridal (St. Lawrence River Institute
of Environmental Sciences) for help with sampling, and to Fida Ahmed
(University of Ottawa) for her assistance in the laboratory. The donation
of the ERE-TK-luc construct from Dr. Farzad Pakdel (Rennes) is acknowledged with gratitude.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.scitotenv.2012.04.069.
References
2006 Community Profiles [internet]. Ottawa (ON): Statistics Canada. c2011 — [cited
2011 Aug 10. Available from: http://www12.statcan.ca/english/census06/data/
profiles/community/Index.cfm?Lang=E.
Ackermann GE, Brombacher E, Fent K. Development of a fish reporter gene system for
the assessment of estrogenic compounds and sewage treatment plant effluents.
Environ Toxicol Chem 2002;21:1864–75.
Aherne GW, Briggs R. The relevance of the presence of certain synthetic steroids in the
aquatic environment. J Pharm Pharmacol 1989;41:735–6.
Aravindakshan J, Paquet V, Gregory M, Dufresne J, Fournier M, Marcogliese DJ, et al.
Consequences of xenoestrogen exposure on male reproductive function in spottail
shiner (Notropis hudsonius). Toxicol Sci 2004;78:156–65.
Atkinson SK, Marlatt VL, Kimpe LE, Lean DRS, Trudeau VL, Blais JM. Environmental factors affecting ultraviolet photodegradation rates and estrogenicity of estrone and
ethinylestradiol in natural waters. Arch Environ Contam Toxicol 2011;60:1–7.
Barel-Cohen K, Shore LS, Shemesh M, Wenzel A, Mueller J, Kronfeld-Schor N. Monitoring of natural and synthetic hormones in a polluted river. J Environ Manage
2006;78:16–23.
Carbella M, Omil F, Lema JM, Llompart M, García-Jares C, Rodríguez I, et al. Behaviour of
pharmaceuticals, cosmetics and hormones in a sewage treatment plant. Water Res
2004;38:2918–26.
Cespedes R, Petrovic M, Raldua D, Saura U, Pina B, Lacorte S, et al. Integrated procedure
for determination of endocrine-disrupting activity in surface waters and sediments
by use of biological technique recombinant yeast assay and chemical analysis by LC
−ESI-MS. Anal Bioanal Chem 2004;378:697–708.
Christiansen LB (University of Southern Denmark, Denmark), Winther-Nielsen M,
Helweg C (DHI, Water & Environment, Denmark). Feminization of fish. Final report. Danish Environmental Protection Agency, Danish Ministry of the Environment; 2002. Environmental project no. 729.
Cicek N, Londry K, Oleszkiewicz JA, Wong D, Lee Y. Removal of selected natural and
synthetic estrogenic compounds in a Canadian full-scale municipal wastewater
treatment plant. Water Environ Res 2007;79(7):795–800.
City of Ottawa. Public Works and Services Department, Utility Services Branch, Wastewater and Drainage Services Division. Robert O. Pickard Environmental Centre annual operations report. Ottawa (ON): City of Ottawa; 2005.. 47 pp.
D'Ascenzo G, Di Corcia A, Gentili A, Mancini R, Mastropasqua R, Nazzari M, et al. Fate of
natural estrogen conjugates in municipal sewage transport and treatment facilities. Sci Total Environ 2003;302:199–209.
de Hoffman E, Stroobant V. Mass spectrometry: principles and applications. John Wiley
and Sons; 2002. 407 pp.
Desbrow C, Routledge EJ, Brighty GC, Sumpter JP, Waldock M. Identification of estrogenic
chemicals in STW effluent. 1. Chemical fractionation and in vitro biological screening.
Environ Sci Technol 1998;32(11):1549–58.
Desforges JP, Peachey BDL, Sanderson PM, White PA, Blais JM. Plasma vitellogenin in
male teleost fish from 43 rivers worldwide is correlated with upstream human
population size. Environ Pollut 2010;158:3279–84.
125
Fernandez MP, Ikonomou MG, Buchanan I. An assessment of estrogenic organic contaminants in Canadian waters. Sci Total Environ 2007;373:250–69.
Gagné F, Marcogliese DJ, Blaise C, Gendron AD. Occurrence of compounds estrogenic to
freshwater mussels in surface waters in an urban area. Environ Toxicol 2001;16:260–8.
Google Earth (version 4.2.0198.2451) [software]. Mountain View, CA: Google Inc.; 2007.
Johnson AC, Sumpter JP. Removal of endocrine-disrupting chemicals in activated
sludge treatment works. Environ Sci Technol 2001;35(24):4697–703.
Johnson AC, Belfroid A, Di Corcia A. Estimating steroid oestrogen inputs into activated
sludge treatment works and observations on their removal from the effluent. Sci
Total Environ 2000;256:163–73.
Johnson AC, Williams RJ, Matthiessen P. The potential steroid hormone contribution of
farm animals to freshwaters, the United Kingdom as a case study. Sci Total Environ
2006;362(1–3):166–78.
Jürgens MD, Holthaus KIE, Johnson AC, Smith JJL, Hetheridge M, Williams RJ. The potential for estradiol and ethinylestradiol degradation in English rivers. Environ
Toxicol Chem 2002;21(3):480–8.
Khanal SK, Xie B, Thomspon M, Sung S, Ong S, VanLeeuwen J. Fate, transport, and biodegradation of natural estrogens in the environment and engineered systems. Environ Sci Technol 2006;40(21):6537–46.
Kirk LA, Tyler CR, Lye CM, Sumpter JP. Changes in estrogenic and androgenic activities
at different stages of treatment in wastewater treatment works. Environ Toxicol
Chem 2001;21:972–9.
Kuch HM, Ballschmiter K. Determination of endocrine-disrupting phenolic compounds
and estrogens in surface and drinking water by HRGC−(NCI)−MS in the picogram
per liter range. Environ Sci Technol 2001;35:3201–6.
Labadie P, Budzinski H. Determination of steroidal hormone profiles along the Jalle
d'Eysines River (near Bordeaux, France). Environ Sci Technol 2005;39(14):5113–20.
Lai KM, Scrimshaw MD, Lester JN. Prediction of the bioaccumulation factors and body
burden of natural and synthetic estrogens in aquatic organisms in the river systems. Sci Total Environ 2002;289:159–68.
Lee HB, Liu D. Degradation of 17β-estradiol and its metabolites by sewage bacteria.
Water Air Soil Pollut 2002;134:353–68.
Lee HB, Peart TE. Determination of 17β-estradiol and its metabolites in sewage effluent
by solid-phase extraction and gas chromatography/mass spectrometry. J AOAC Int
1998;81:1209–16.
Lishman L, Smyth SA, Sarafin K, Kleywegt S, Toito J, Peart T, et al. Occurrence and reductions
of pharmaceuticals and personal care products and estrogens by municipal wastewater
treatment plants in Ontario, Canada. Sci Total Environ 2006;367(2–3):544–58.
National Climate Data and Information Archive. [Internet]. Fredericton (NB): Environment
Canada. [cited 2011 Aug 10]. Available from:http://www.climate.weatheroffice.ec.gc.
ca/climateData/canada_e.html2011.
Ontario Centre for Municipal Best Practices. Best practice summary report. Water and
wastewater alternative sources of energy. Case study of Ottawa. Toronto (ON): Association of Municipalities of Ontario; 2006. 5 pp.
Salste L, Leskinen P, Virta M, Kronberg L. Determination of estrogens and estrogenic activity in wastewater effluent by chemical analysis and the bioluminescent yeast
assay. Sci Total Environ 2007;378:343–51.
Schlüsener MP, Bester K. Behaviour of steroid hormones and conjugates during wastewater treatment — a comparison of three sewage treatment plants. Clean
2008;36(1):25–33.
Servos MR, Bennie DT, Burnison BK, Jurkovic A, McInnis R, Neheli T, et al. Distribution
of estrogens, 17β-estradiol and estrone, in Canadian municipal wastewater treatment plants. Sci Total Environ 2005;336(1–3):155–70.
Shiwei J, Yang F, Liao T, Hui Y, Xu Y. Seasonal variation of estrogenic compounds and
their estrogenicities in influent and effluent from a municipal sewage treatment
plant in China. Environ Toxicol Chem 2008;27(1):146–53.
Sierra Legal Defence Fund. The national sewage report card (number three): grading
the sewage treatment of 22 Canadian cities. Toronto (ON): Sierra Legal Defence
Fund; 2004. 68 pp.
Sierra Legal Defence Fund. The Great Lakes sewage report card. Toronto (ON): Sierra
Legal Defence Fund; 2006. 57 pp.
Suidan MT, Esperanza M, McCauley P, Brenner RC, Venosa AD. Challenges in biodegradation of trace organic contaminants — gasoline oxygenates and sex hormones.
Water Environ Res 2005;77(1):4-11.
Ternes TA, Stumpt M, Mueller J, Harbere K, Wilken RD, Servos M. Behaviour and occurrence of estrogens in municipal sewage treatment plants — I. Investigations in
Germany, Canada and Brazil. Sci Total Environ 1999a;225:81–90.
Ternes TA, Kreckel P, Mueller J. Behaviour and occurrence of estrogens in municipal
sewage treatment plants — II. Aerobic batch experiments with activated sludge.
Sci Total Environ 1999b;225:91–9.
Thorpe KL, Gross-Sorokin M, Johnson I, Brighty G, Tyler CR. An assessment of the model of
concentration addition for predicting the estrogenic activity of chemical mixtures in
wastewater treatment works effluents. Environ Health Perspect 2006;114:90–7.
Trudeau VL, Heyne B, Blais JM, Temussi F, Atkinson SK, Pakdel F, et al. Lumiestrone is photochemically derived from estrone and may be released to the environment without
detection. Front Exp Endocrinol 2011. http://dx.doi.org/10.3389/fendo.2011.00083/full.
United States Environmental Protection Agency. U.S. Code of Federal Regulations, title
40, part 136, appendix B. Washington (VA): US EPA; 1993.
United States Environmental Protection Agency. Lake Ontario lakewide management
plan status 2008. Chicago (Il): US EPA; 2008. 130 pp.
Van den Belt K, Berckmans P, Vangenechten C, Verheyen R, Witters H. Comparative
study on the in vitro/in vivo estrogenic potencies of 17β-estradiol, estrone, 17αethynylestradiol and nonylphenol. Aquat Toxicol 2004;66:183–95.
Williams RJ, Johnson AC, Smith JJL, Kanda R. Steroid estrogens profiles along river
stretches arising from sewage treatment works discharges. Environ Sci Technol
2003;37:1744–50.