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: kmyleung@hku.hk; zhougj01@gmail.com

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.

6

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).

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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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