Retinoids and oestrogenic endocrine disrupting chemicals in saline sewage
treatment plants: Removal efficiencies and ecological risks to marine organisms
Guang-Jie Zhoua*, Xiao-Yan Lib, Kenneth Mei Yee Leunga,c*
a The Swire Institute of Marine Science and School of Biological Sciences, The
University of Hong Kong, Pokfulam, Hong Kong, China
b Environmental Engineering Research Centre, Department of Civil Engineering, The
University of Hong Kong, Pokfulam, Hong Kong, China
c State Key Laboratory of Marine Pollution (City University of Hong Kong), Tat Chee
Avenue, Kowloon, Hong Kong, China
*Co-corresponding authors.
Co-corresponding authors: Professor Kenneth M. Y. Leung; Dr G.J. Zhou
Corresponding address: School of Biological Sciences, The University of Hong
Kong, Pokfulam, Hong Kong, China
Corresponding tel.: +852 22990607
Corresponding fax: +852 25176082
Corresponding emails: [email protected]; [email protected]
1
Chemicals and materials
High-purity standards of all-trans-RA (at-RA), 13-cis-RA (13c-RA), 9-cis-RA (9c-
RA), acitretin and estrone (E1) were purchased from Sigma-Aldrich (St. Louis, USA),
and all-trans-4-oxo-RA (at-4-oxo-RA), 13-cis-4-oxo-RA (13c-4-oxo-RA), 9-cis-4-
oxo-RA (9c-4-oxo-RA) and at-RA-d5 were purchased from Toronto Research
Chemicals (Toronto, Ontario, Canada). 13C12-TCS, 13C6-TCC and E1-d4 were
purchased from Cambridge Isotope Laboratories, Inc. (Massachusetts, USA). 4-
nonylphenol (4-NP), 4-n-NP and triclocarban (TCC) were purchased from Dr.
Ehrenstorfer GmbH (Germany), and 4-t-octylphenol (4-t-OP), bisphenol-A (BPA) and
BPA d16 were purchased from Supelco (USA). Diethylstilbestrol (DES) was
purchased from Chiron (Norway), and triclosan (TCS) was purchased from Alfa Aesar
(USA). The chemical information for the target compounds is presented in Table S1.
HPLC grade acetic acid and formic acid were purchased from Sigma-Aldrich (St.
Louis, USA). HPLC grade methanol, acetonitrile, hexane and ethyl acetate were
purchased from Tedia (Fairfield, OH, USA). Oasis HLB cartridges (6 mL, 500 mg)
were obtained from Waters Corporation (Milford, MA, USA). Ammonium acetate
(≥98%), anhydrous sodium sulphate (≥99%) and silica gel were purchased from
Sigma-Aldrich (St. Louis, USA), and glass fibre filters (GF/F, pore size 0.7 μm) were
supplied by Whatman (Maidstone, England). Ultrapure water was obtained from a
Milli-Q synthesis water purification system (Tin Hang Technology Limited, Hong
Kong).
Sample collection
Three sewage treatment plants (STPs) in Hong Kong, namely, the Shatin STP, the
2
Stanley STP and the Stonecutters Island STP, were chosen in this study to investigate
the concentrations of retinoids and EDCs in each stage of the wastewater treatment
process and their respective adjacent receiving seawaters (Fig. S1). The Shatin STP is
the largest secondary biological sewage treatment plant in Hong Kong, serving a
population of 600,000 inhabitants in the Shatin and Ma On Shan Districts and treating
253,000 m3/day of sewage (Table S2; HKDSD, 2016). The wastewater treatment
process in the Shatin STP consists of preliminary treatment (screening and degritting),
primary sedimentation, and secondary biological treatment followed by final
sedimentation. The secondary effluent is further treated with UV irradiation prior to
discharge as the final effluent to the adjacent coastal marine environment. The
Stanley STP, which is the first STP built in caverns in Hong Kong, is a secondary
biological sewage treatment plant that receives approximately 8200 m3/day of sewage
generated by 27,000 residents of Stanley (Table S2; HKDSD, 2016). The wastewater
treatment process in the Stanley STP is similar to that of the Shatin STP, except that
the effluent is disinfected by chlorination prior to discharge. The Stonecutters Island
STP is the largest sewage treatment plant using the chemically enhanced primary
treatment (CEPT) in the world, serving a population of 3,500,000 inhabitants and
treating 1,961,000 m3/day of sewage collected by the seven preliminary treatment
works in the main urban areas of Kowloon and Northeast Hong Kong Island (Table
S2; HKDSD, 2016). The wastewater treatment process in the Stonecutters Island STP
includes screening and FeCl3 treatment followed by sedimentation. The wastewater
from the sedimentation tank is further treated by chlorination and dechlorination prior
to the discharge to adjacent seawater. Basic information and process flow charts of
the three STPs are presented in Table S2 and Fig. 1.
3
Samples of seawater, wastewater and dewatered sludge (n = 3) were collected in
November 2016 from each of the sampling points in the three sewage treatment plants
(Fig. 1). The collected seawater or wastewater samples were adjusted to pH 3 using 4
M H2SO4, and methanol was added to the samples (5% v/v) to inhibit microbial
activity; then, the samples were transported in a cooler back to the laboratory and
were stored in the dark at 4 °C. All of the samples were collected in amber bottles
and were extracted within 24 h after the collection using solid phase extraction (SPE).
The dewatered sludge samples were collected in glass bottles, freeze-dried after their
arrival in the laboratory, and kept in a freezer at –18 °C until extraction.
Extraction and cleanup
Extractions of the samples were carried out following the method described by Chen
et al. (2010) and Wu et al. (2010) with modifications, using the method described in
detail below. After filtration with glass fibre filters (Whatman GF/F, 0.7 μm, UK),
water samples (50 mL for influent, 1 L for seawater and 100 mL for others) spiked
with internal standards (75 ng each) were passed through the Oasis HLB cartridges (6
mL, 500 mg, Waters) that were pre-conditioned with ethyl acetate (6 mL), methanol
(6 mL) and H2O (12 mL) in sequence. The water samples were introduced to the
cartridges at a flow rate of 5–8 mL min-1. The sample bottle was rinsed twice with
two aliquots (50 mL) of 5% (v/v) methanol in ultrapure water that passed through the
cartridges. The cartridges were then dried with nitrogen gas for at least 1 h. The
target compounds were eluted with ethyl acetate containing 0.5% formic acid (10
mL), and were allowed to drip through the cartridges under action of gravity. The
extracts were evaporated to near dryness under a gentle stream of nitrogen at room
temperature, were redissolved in hexane (1 mL), and then were purified using silica
4
gel columns (23 cm* 0.6 cm i.d.) consisting of anhydrous sodium sulphate (0.5 cm,
on top), silica gel (1 g, in middle) and glass wool (on bottom). Each extract in hexane
was loaded onto the silica gel column that was preconditioned sequentially with
methanol (4 mL), ethyl acetate (4 mL) and hexane (6 mL). After the cartridge was
rinsed with hexane (6 mL), the target compounds were eluted with ethyl
acetate/methanol (6 mL, 90:10, v/v). The eluate was then dried under a gentle
nitrogen stream and reconstituted in acetonitrile (150 μL). The sample was stored in 2
mL amber glass vials at –18 °C until analysis.
Freeze-dried sludge samples (0.1 g each) spiked with internal standards (75 ng each)
were extracted with ethyl acetate (5 mL) in an ultrasonic bath for 10 min and then
were centrifuged at 2375 g for 5 min. The supernatant was transferred into a 15 mL
glass tube. The extraction process was repeated twice using 5 mL and 2 mL of ethyl
acetate. Then, the 12 mL of extract from each sample was combined, evaporated to
near dryness under a gentle stream of nitrogen at room temperature, and purified by
the cleanup method described above prior to the injection to the HPLC-MS/MS for
the analysis of the target chemicals.
Instrumental Analysis
The target compounds were separated into three groups (RAs, 4-oxo-RAs and EDCs)
and were analysed by HPLC-MS/MS using an Agilent 1290 HPLC coupled to a 3200
QTRAP mass spectrometer equipped with Analyst 1.5.2 data-processing software.
Chromatographic separation of each group was performed on an Agilent Zorbax
RRHD Eclipse Plus C-18 (100 mm × 2.1 mm, 1.8 µm) with its corresponding guard
column (5 mm × 2.1 mm, 1.8 µm) at a flow rate of 0.3 mL/min at 40 °C. The mobile
5
phase for the analysis of RAs consisted of (A) ultrapure water containing 0.01%
acetic acid, and (B) acetonitrile. The gradient programme was as follows: 0–0.5 min,
90–25% A; 0.5–9 min, 25–23% A; 9–10 min 0% A; 10–12 min, re-equilibration with
90% A. The mobile phase for the analysis of 4-oxo-RAs consisted of (A) ultrapure
water containing 2 mM ammonium acetate, and (B) acetonitrile. The gradient
programme was as follows: 0–0.5 min, 90–50% A; 0.5–4 min, 50–48% A; 4–6 min
0% A; 6–8 min, re-equilibration with 90% A. The mobile phase for the analysis of
EDCs consisted of (A) ultrapure water, and (B) acetonitrile. The gradient programme
was as follows: 0–0.5 min, 90–70% A; 0.5–12 min, 70–30% A; 12–17 min, 30–0% A;
17–18 min 0% A; 18–20 min, re-equilibration with 90% A. Injection volumes for
RAs and 4-oxo-RAs were 5 µL, and the volumes for EDCs were 10 µL. HPLC-
MS/MS chromatograms of RAs, 4-oxo-RAs and EDCs in standard are shown in Fig.
S2.
An electrospray ionization (ESI) source was used in negative ion mode, and multiple
reaction monitoring (MRM) mode was used to analyse the target compounds.
Source-dependent parameters, including curtain gas (CUR), collision gas (CAD),
ionSpray voltage (IS), temperature (TEM), ion source gas 1 (GS1) and ion source gas
2 (GS2) were optimized in flow injection analysis (FIA) at the optimal LC flow and
mobile phase composition. The optimized parameters are listed in Table S3.
Compound-dependent parameters including the declustering potential (DP), entrance
potential (EP), collision energy (CE), collision cell exit potential (CXP), and collision
cell entrance potential (CEP) were optimized automatically using the infusion
method, and the optimized parameters are also listed in Table S3. Nitrogen gas was
used as the drying and collision gas.
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Quality assurance and quality control
All equipment was rinsed with methanol to avoid sample contamination. An
operational blank was examined with each batch of analyses in the same manner as
the samples. At-RA-d5, acitretin, 4-n-nonylphenol, bisphenol-A d16, oestrogen-d4,
13C12-triclosan and 13C6-triclocarban were used as the internal standards for the
quantification of retinoids and EDCs (Table S1). The relative recoveries for the
retinoids and EDCs spiked into the wastewater samples ranged from 65–160% and
100–154%, respectively, and those in the sludge samples ranged from 70–158% and
63–182%, respectively (Table 1). Limits of quantification (LOQs) for retinoids and
EDCs in the wastewater were 0.60–2.8 ng/L and 0.28–21 ng/L, respectively, and the
LOQs in the sludge were 0.56–2.8 ng/g and 1.2–31 ng/g, respectively (Table 1). To
prevent photodegradation and photoisomerization of the retinoids and EDCs, the
samples were collected using amber bottles, and experiments were carried out in dark
conditions whenever possible. All cartridges used for solid-phase extraction and
cleanup were wrapped in aluminium foil.
Mass balance analysis
Mass balance analysis was used to estimate the mass flow of the chemicals entering
and leaving the sewage treatment plant in both wastewater and sludge forms. The loss
mass of a chemical (Mloss, g/d), mainly caused by the contribution of the degradation
process, during the entire STP treatment was calculated using the following equation:
Mloss = Minfluent – Meffluent – Msludge, (1)
where Minfluent, Meffluent and Msludge are the mass loads (g/d) of the chemical in the
influent, effluent and dewatered sludge, respectively.
7
According to equation (1), the percentage of mass loss of a chemical was calculated
using the following equation:
Mloss % = (Minfluent – Meffluent – Msludge)/Minfluent × 100%, (2)
Then, the mass percentages for each chemical in the effluent and dewatered sludge
were calculated as Meffluent/Minfluent% and Msludge/Minfluent%, respectively.
The aqueous phase removal efficiency (R%) of a chemical in each treatment process
of the STPs was calculated using the following equation:
Raqueous% = (Cin – Cout)/Cin × 100%, (3)
where Cin and Cout are the input and output concentrations of the chemical for the
given treatment process. When this value is less than the limit of quantification
(LOQ), the LOQ value divided by two is used for the calculation.
8
Table S1. Detail information for the retinoids and endocrine disrupting chemicals including abbreviation (Abbr.), CAS number (CAS
no.), n-octanol/water partition coefficient (Kow), molecular formula, molecular weight (M.W.), internal standard and purpose of use.
Compound Abbr. CAS no. Log Kow Molecular formula M.W. Internal standard Purpose of useRetinoids
All-trans-retinoic acid At-RA 302-79-4 6.30 C20H28O2300.4
At-trans-retinoic acid-d5
Medicine/Natural metabolite
13-cis-retinoic acid 13c-RA 4759-48-2 6.30 C20H28O2 300.4
At-trans-retinoic acid-d5
Medicine/Natural metabolite
9-cis-retinoic acid 9c-RA 5300-03-8 - C20H28O2 300.4
At-trans-retinoic acid-d5
Medicine/Natural metabolite
All-trans-4-oxo-retinoic acid At-4-oxo-RA 38030-57-8 - C20H26O3
314.4 Acitretin Natural metabolite
13-cis-4-oxo-retinoic acid 13c-4-oxo-RA 71748-58-8 - C20H26O3 314.
4 Acitretin Natural metabolite
9-cis-4-oxo-retinoic acid 9c-4-oxo-RA 150737-18-1
- C20H26O3 314.4 Acitretin Natural metabolite
Endocrine disrupting chemicals
4-Nonylphenol 4-NP 84852-15-3 5.76 C15H24O220.4 4-n-Nonylphenol Industrial chemical
4-tert-Octylphenol 4-t-OP 140-66-9 4.12 C14H22O206.3 4-n-Nonylphenol Industrial chemical
Bisphenol-A BPA 80-05-7 3.32 C15H16O2228.3 Bisphenol-A d16 Industrial chemical
Estrone E1 53-16-7 3.43 C18H22O2270.4 Estrone-d4 Natural oestrogen
Diethylstilbestrol DES 56-53-1 5.07 C18H20O2268.4 Estrone-d4 Synthetic oestrogen
Triclosan TCS 3380-34-5 4.7 C12H7Cl3O2289.5
13C12-Triclosan Antiseptic and disinfectant
Triclocarban TCC 101-20-2 4.9 C13H9C13N2O315.6
13C6-Triclocarban Antiseptic and disinfectant
9
10
Table S2 Basic parameters of the three sewage treatment plants (STPs) in Hong Kong, and the physiochemical parameters of their
effluents. The water quality data corresponding to each sampling date (2016–11–14 for Stonecutters Island STP, 2016–11–21 for
Shatin STP, and 2016–11–28 for Stanley STP) was collected from the Hong Kong Drainage Services Department. If the data are not
available at the sampling dates, the average values for November 2016 were used. Please see the website below for more details
(http://www.dsd.gov.hk/SC/Files/sewerage/our_sewage_treatment_facilities/effluent_quality_of_8_major_works/
2016/201611_STW_effluen.pdf). CEPT, chemically enhanced primary treatment; BOD5, 5d biochemical oxygen demand; TSS, total
suspended solids; NOx–N, NO3–N + NO2–N; Total–N, total nitrogen; TRC, total residual chlorine. 24 h composite effluent samples
were used for determining BOD5, TSS, NH3–N, NOx–N and Total–N, but grab effluent samples were used for determining TRC and E.
coli.
Shatin STP Stanley STP Stonecutters Island STPSewage treatment facilities Secondary biological treatment Secondary biological treatment CEPTService population (103) 600 27 3500Design flow (103 m3/d) 340 11.6 4000Daily flow (103 m3/d) 253 8.2 1961Hydraulic retention time (hour) 20 13.6 1.5Dewatered sludge (Tonne/d) 104 2.4 755BOD5 (mg O2/L) <5 <3 78TSS (mg/L) 12 <4 44NH3–N (mg/L) 5.9 <0.48NOx–N (mg/L) <3.8Total–N (mg/L) 9.8
11
TRC (mg/L) <0.1E. coli (count/100 mL) 320 41 29000Table S3 Target chemicals and their optimized parameters in HPLC-MS/MS in the negative ion mode.
Compound SupplierRT
(min)
Precursor
ion
(m/z)
Product ions
(m/z)
CU
R
(psi)
CADIS
(V)
TE
M
(°C)
GS1
(psi
)
GS2
(psi
)
DP
(V)
EP
(V)
CE
(eV)
CX
P
(V)
CE
P
(V)
Retinoids
At-RA Sigma-Aldrich (USA) 8.418 299.1 255.1 118.9 20Mediu
m-4500 750 20 20 -45 -9.5
-20/-
35-2 -14
13c-RA Sigma-Aldrich (USA) 7.829 299.1 255.1 118.9 20Mediu
m-4500 750 20 20 -45 -9.5
-20/-
35-2 -14
9c-RA Sigma-Aldrich (USA) 8.186 299.1 255.1 118.9 20Mediu
m-4500 750 20 20 -45 -9.5
-20/-
35-2 -14
At-4-oxo-RAToronto Research Chemicals
(Canada)2.744 313.1 269.2 119.1 20
Mediu
m-4500 750 25 40 -50 -3.5
-25/-
25-2 -20
13c-4-oxo-RAToronto Research Chemicals
(Canada)3.147 313.1 269.2 119.1 20
Mediu
m-4500 750 25 40 -50 -3.5
-25/-
25-2 -20
9c-4-oxo-RAToronto Research Chemicals
(Canada)3.085 313.1 269.2 119.1 20
Mediu
m-4500 750 25 40 -50 -3.5
-25/-
25-2 -20
RA-d5 (I.S.)Toronto Research Chemicals
(Canada)7.744 304.1 260.2 120.1 20
Mediu
m-4500 750 20 30 -50
-
10.5
-25/-
35-4 -16
Acitretin (I.S.) Sigma-Aldrich (USA) 5.356 325.0 266.2 281.3 20Mediu
m-4500 750 25 20 -40 -10
-20/-
20-4 -14
Endocrine disrupting chemicals
4-NP Dr. Ehrenstorfer GmbH (Germany) 14.031 219.0 133.1 119.0 10 High -4500 700 40 60 -65 -4.5-36/-
480 -12
4-t-OP Supelco (USA) 12.445 205.0 133.0 116.8 10Mediu
m-4500 550 20 30 -60 -9
-34/-
90-2 -14
12
BPA Supelco (USA) 5.584 227.0 211.9 133.0 10 High -4500 700 50 30 -65-
10.5
-20/-
28-2 -20
E1 Sigma-Aldrich (USA) 7.047 269.0 145.0 143.0 10 High -4000 750 30 20-
110
-
10.5
-54/-
700 -20
DES Chiron (Norway) 7.708 267.0 236.9 250.9 10 High -4500 450 40 50 -75 -4.5-26/-
26-2 -18
4-n-NP (I.S.) Dr. Ehrenstorfer GmbH (Germany) 15.290 219.1 106.0 119.0 20Mediu
m-4500 700 50 20 -70
-
10.5
-28/-
560 -16
BPA d16 (I.S.) Supelco (USA) 5.445 241.0 222.9 142.0 10Mediu
m-4000 600 30 30
-
100-4.5
-20/-
36-2 -58
E1-d4 (I.S.)Cambridge Isotope Laboratories
Incorporation (Massachusetts, USA)7.075 273.0 147.0 161.0 10 High -4500 550 20 30
-
290
-
10.5
-52/-
480 -14
TCS Alfa Aesar (USA) 11.949 286.7 35.1 20 Low -4500 600 40 40 -40 -4.5 -24 -4 -14
TCC Dr. Ehrenstorfer GmbH (Germany) 11.765 312.9 159.9 125.9 20Mediu
m-4500 650 50 40 -60 -11
-16/-
300 -34
13C12-TCS (I.S.)Cambridge Isotope Laboratories
Incorporation (Massachusetts, USA)11.920 298.8 35.1 10 Low -4000 700 20 30 -30
-
10.5-28 -4 -30
13C6-TCC (I.S.)Cambridge Isotope Laboratories
Incorporation (Massachusetts, USA)11.770 318.9 159.9 132.0 10
Mediu
m-3500 700 30 30 -60 -4.5
-22/-
320 -22
(I.S.) internal standard; (RT) retention time (min); (CRU) curtain gas; (CAD) collision gas; (IS) ionSpray voltage; (TEM) temperature; (GS1) ion source gas 1; (GS2) ion source
gas 2; (DP) declustering potential; (EP) entrance potential; (CE) collision energy; (CXP) collision cell exit potential; (CEP) collision cell entrance potential. Please see Table S1 for
the full name of each compound.
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Table S4 Removal efficiencies (%, mean ± SD, n = 3) of the retinoids and endocrine disrupting chemicals (EDCs) for each treatment
stage of the Shatin sewage treatment plant.
Compound Primary treatment Biological treatment Final sedimentation UV treatment Total removal efficiencyRetinoidsAt-RA 50 ± 0 -134 ± 104 -30 ± 12 67 ± 0 50 ± 013c-RA 76 ± 0 0 ± 0 0 ± 0 0 ± 0 76 ± 09c-RA - - - - -At-4-oxo-RA -42 ± 7 86 ± 2 44 ± 3 -11 ± 45 87 ± 513c-4-oxo-RA -837 ± 199 64 ± 5 -35 ± 27 89 ± 0 50 ± 09c-4-oxo-RA - - - - -Total retinoids -53 ± 13 75 ± 2 0 ± 12 55 ± 10 82 ± 4Endocrine disrupting chemicals4-NP -3 ± 11 -14 ± 20 7 ± 13 -3 ± 27 -13 ± 294-t-OP 61 ± 33 -49 ± 54 -19 ± 94 50 ± 10 66 ± 7BPA 23 ± 14 36 ± 6 4 ± 9 24 ± 7 64 ± 3E1 - 100 ± 0 - - -DES 50 ± 0 0 ± 0 0 ± 0 0 ± 0 50 ± 0TCS -92 ± 25 24 ± 31 31 ± 12 1 ± 9 1 ± 9TCC -51 ± 10 -15 ± 16 -56 ± 10 15 ± 9 -131 ± 24Total EDCs 14 ± 14 4 ± 13 4 ± 18 13 ± 16 31 ± 13– Not available: the input concentration of a chemical for the treatment stage is zero, leading to failed calculation.
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Table S5 Removal efficiencies (%, mean ± SD, n = 3) of the retinoids and endocrine disrupting chemicals (EDCs) for each treatment
stage of the Stanley sewage treatment plant.
Compound Biological treatment Final sedimentation Chlorination Total removal efficiencyRetinoidsAt-RA -55 ± 14 68 ± 0 0 ± 0 50 ± 013c-RA 50 ± 0 0 ± 0 0 ± 0 50 ± 09c-RA - - - -At-4-oxo-RA 87 ± 0 0 ± 0 0 ± 0 87 ± 013c-4-oxo-RA - 59 ± 0 -142 ± 126 -9c-4-oxo-RA - - - -Total retinoids 24 ± 6 47 ± 0 -30 ± 27 48 ± 11Endocrine disrupting chemicals4-NP 58 ± 9 67 ± 5 8 ± 32 87 ± 44-t-OP -195 ± 88 93 ± 1 31 ± 32 86 ± 6BPA -14 ± 18 45 ± 5 -41 ± 4 11 ± 2E1 -464 ± 130 26 ± 19 38 ± 26 -161 ± 109DES 50 ± 0 0 ± 0 0 ± 0 50 ± 0TCS -324 ± 281 73 ± 8 -81 ± 50 -104 ± 56TCC -99 ± 17 -24 ± 24 27 ± 8 -80 ± 21Total EDCs 32 ± 12 69 ± 4 -1 ± 20 79 ± 4– Not available: the input concentration of a chemical for the treatment stage is zero, leading to failed calculation.
15
Table S6 Removal efficiencies (%, mean ± SD, n = 3) of the retinoids and endocrine disrupting chemicals (EDCs) for each treatment
stage of the Stonecutters Island sewage treatment plant.
Compound Chemical treatment Chlorination Dechlorination Total removal efficiencyRetinoidsAt-RA 50 ± 0 0 ± 0 0 ± 0 50 ± 013c-RA 50 ± 0 0 ± 0 0 ± 0 50 ± 09c-RA - - - -At-4-oxo-RA 53 ± 4 -29 ± 9 -7 ± 26 34 ± 1613c-4-oxo-RA 21 ± 26 -40 ± 7 54 ± 8 49 ± 89c-4-oxo-RA - - - -Total retinoids 45 ± 5 -26 ± 3 15 ± 11 41 ± 7Endocrine disrupting chemicals4-NP 41 ± 22 -29 ± 24 -7 ± 27 18 ± 214-t-OP 5 ± 73 23 ± 57 -86 ± 132 -36 ± 96BPA 64 ± 5 -27 ± 13 47 ± 4 76 ± 2E1 50 ± 0 0 ± 0 0 ± 0 50 ± 0DES 32 ± 7 29 ± 32 -15 ± 34 44 ± 17TCS 24 ± 8 20 ± 6 -73 ± 85 -5 ± 51TCC 8 ± 2 24 ± 9 12 ± 11 38 ± 8Total EDCs 54 ± 7 -19 ± 14 14 ± 17 53 ± 9– Not available: the input concentration of a chemical for the treatment stage is zero, leading to failed calculation.
Table S7 Mass proportions (%, mean ± SD, n = 3) of the retinoids and endocrine disrupting chemicals (EDCs) in the effluent,
dewatered sludge and total loss relative to the calculated initial mass loading (100%) in three sewage treatment plants (STPs).
16
CompoundShatin STP Stanley STP Stonecutters Island STP
Effluent (%)
Sludge (%)
Loss (%)
Effluent (%)
Sludge (%)
Loss (%)
Effluent (%)
Sludge (%)
Loss (%)
RetinoidsAt-RA 50 33 17 50 57 -7 50 162 -11213c-RA 24 25 50 50 50 0 50 68 -189c-RA - - - - - - - - -At-4-oxo-RA 13 0 87 13 0 87 66 0 3413c-4-oxo-RA 50 0 50
- - -51 0 49
9c-4-oxo-RA - - - - - - - - -Total Retinoids
18 5 77 52 27 21 59 25 16
Endocrine disrupting chemicals4-NP 113 221 -234 13 7 81 82 11 84-t-OP 34 152 -86 14 5 81 136 45 -81BPA 36 2 62 89 3 8 24 3 73E1 - - - 261 5 -167 50 7 43DES 50 0 50 50 129 -79 56 4 40TCS 99 78 -77 204 207 -311 105 163 -168TCC 231 1258 -1388 180 1000 -1079 62 3242 -3203Total EDCs 69 130 -99 21 12 67 47 34 18– Not available: the input concentration of a chemical in the influent is zero, leading to failed calculation.
Table S8 Concentrations (ng/L) of the endocrine disrupting chemicals (EDCs) in surface seawaters worldwide. The sampling sites
corresponding to the codes are shown in Fig. S3. LOD denotes limit of detection.
Site Code
4-NP 4-t-OP BPA E1 DES TCS TCC References
17
Coast, Hong Kong 1 45–160 13–20 9–71 <LOD1.0–3.9 1.7–2.8 0.35–3.1 This study
Coast near PRE, China 1 31–1777 11–777 Liu et al., 2010Coastal seawaters, Hong Kong 1 77–1354 4.4–114 Xu et al., 2018Marine protected areas, Hong Kong 1 13–188 1.3–18 3.4–20 <LOD–1.0 Xu et al., 2016Marine Reserve, Hong Kong 1 92–474 14–207 Xu et al., 2015Marine Reserve, Hong Kong 1 61–497 11–408 Xu et al., 2014aPearl River Estuary (PRE), China 1 <20–39 <2–9 Chen et al., 2006PRE, China 1 <LOD–163 <LOD–178 Xu et al., 2014bSeawaters, Hong Kong 1 <10–270 Kueh and Lam, 2008Seawaters, Hong Kong 1 16–99 Wu et al., 2007Jiulong River Estuary, China 2 <LOD–925 <LOD–97 <LOD–47 Sun et al., 2016Jiulong River Estuary, China 2 2.6–27 0.38–5.8 Lv et al., 2014Coast, Taiwan 3 290–370 61–66 Cheng et al., 2006Danshuei River Estuary, Taiwan 4 4.3–10 <LOD Shen et al., 2012Jiaozhou Bay, China 5 20–269 1.2–16 1.5–93 Fu et al., 2007Qingdao Port, China 5 12–71 <LOD–28 Wang et al., 2013Daliao River Estuary, China 6 26–675 <LOD–11 13–137 Li et al., 2013Saemangeum Bay, Korea 7 <LOD–247 Li et al., 2005Masan Bay, Korea 8 9.7–207 Li et al., 2008Suruga Bay, Japan 9 28–276 <0.80 3.6–1070 <1.0–9.2 Hashimoto et al., 2007Tokyo Bay, Japan 10 30–104 20–30 5.8–32 Hashimoto et al., 2005Bays, Philippines 11 <LOD–38 <LOD–181 Santiago and Kwan, 2007
Coast, Singapore 12 20–2760<LOD–800
<LOD–2470 Basheer et al., 2004
Coast, Singapore 12 <LOD–694 <LOD–11 <LOD–11 Bayen et al., 2013Coast, Antarctic 13 <LOD 0.30–1.8 <1.3–30 <7.0 Emnet et al., 2015Todos os Santos Bay, Brazil 14 <LOD <LOD–77 <LOD Lisboa et al., 2013Coast, California, USA 15 <LOD–230 <LOD–42 <LOD <LOD <LOD–6.1 Vidal-Dorsch et al., 2012San Francisco Bay, USA 16 <LOD–73 <LOD Klosterhaus et al., 2013
Looe Key, USA 17 <LOD<LOD–0.88 <LOD Singh et al., 2010
Charleston Harbor, USA 18 0.50 Sapozhnikova et al., 2011Charleston Harbor, USA 18 <LOD 12–39 2.9–3.8 Hedgespeth et al., 2012Charleston Harbor, USA 18 4.9–14 Fair et al., 2009Hudson River Estuary, USA 19 1.0–3.0 Wilson et al., 2009Jamaica Bay, USA 19 77–416 1.6–8.3 Ferguson et al., 2001Acushnet River Estuary, USA 20 0.78–1.2 Zuo et al., 2006
18
Narragansett Bay, USA 20 10–75 3.5–11 <LOD Sacks and Lohmann, 2011Massachusetts Bay, USA 21 0.086–0.52 Griffith et al., 2016Halifax Harbour, Canada 22 <LOD–2.6 Robinson et al., 2009Halifax Harbour, Canada 22 4.0–6.6 <LOD Saravanabhavan et al., 2009St. John’s Harbor, Canada 23 1.4–1.5 <LOD Saravanabhavan et al., 2009Douro River Estuary, Portugal 24 11–13 19–27 43–57 0.51–2.0 Rocha et al., 2011Mondego River Estuary, Portugal 24 <LOD <LOD–880 <LOD Ribeiro et al., 2009Ria de Aveiro Sea, Portugal 24 <29–60 <1.2–1.5 <1.1 Jonkers et al., 2010Estuary, Portugal 25 <LOD Neng and Nogueira, 2012Guadalete River Estuary, Spain 26 27–310 Pintado-Herrera et al., 2014
Coast, Spain 27<LOD–4100 Petrovic et al., 2002
NW Mediterranean Sea, Spain 28 1.2–689 1.4–198 Sánchez-Avila et al., 2012Cantabrian Sea, Spain 29 4.3–5999 0.60–740 Sánchez-Avila et al., 2013
Northwest Coast, Spain 30 <30–337 <8–72 <20–146Salgueiro-González et al., 2015
Coast, Ireland 31<LOD–0.76 Ronan and McHugh, 2013
Estuaries, UK 32 100–2600 Blackburn et al., 1999Scheldt Estuary, Netherlands 33 0.37–10 Noppe et al., 2007Estuaries, Netherlands 34 31–934 Jonkers et al., 2003Estuaries, Netherlands 34 <LOD–330 Belfroid et al., 2002
North Sea 35 <LOD<LOD–0.070 Brumovský et al., 2016
Bight, Germany 360.0080–6.9 Xie et al., 2008
North Sea, Germany 36 0.30–84 0.020–18 <LOD–249 Heemken et al., 2001Baltic Sea, Germany 37 2.5–14 0.11–0.60 0.22–5.4 0.10–0.53 Beck et al., 2005Baltic Sea, Poland 38 13–133 <5–66 30–48 Staniszewska et al., 2014Seawaters, Norway 39 <LOD Weigel et al., 2004Venice Lagoon, Italy 40 <LOD–211 <LOD–145 <LOD–10 Pojana et al., 2007Krya River Estuary, Croatia 41 <20–1200 Kveštak and Ahel,1994Thermaikos Gulf, Greece 42 22–201 1.7–18 11–52 <LOD Arditsoglou and Voutsa, 2012Kuwait Bay, Kuwait 43 30 Smith et al., 2015
19
Fig. S1 Locations of the three sewage treatment plants (STPs) (i.e., Shatin STP,
Stanley STP and Stonecutters Island STP, red dots) and the receiving seawaters (blue
dots) in Hong Kong.
20
(A)
(B)
Fig. S2 Extracted ion chromatograms (EIC) of the quantitative ions for (A) retinoic
acids (RAs) and their internal standard RA-d5, (B) the metabolites of RAs, and their
internal standard acitretin, and (C) endocrine disrupting chemicals (EDCs) and their
internal standards in chemical standard solutions.
21
(C)
Fig. S2 (Cont’) Extracted ion chromatograms (EIC) of the quantitative ions for (A)
retinoic acids (RAs) and their internal standard RA-d5, (B) the metabolites of RAs,
and their internal standard acitretin, and (C) endocrine disrupting chemicals (EDCs)
and their internal standards in chemical standard solutions.
22
Fig. S3 Locations for the sampling for endocrine disrupting chemicals (EDCs) in surface seawaters worldwide. The concentrations of EDCs for
23
each sampling site are shown in Table S8.
24
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