at SciVerse ScienceDirect

Environmental Pollution 165 (2012) 241e249

Contents lists available

Environmental Pollution

journal homepage: www.elsevier .com/locate/envpol

Monitoring of selected estrogenic compounds and estrogenic activity in surfacewater and sediment of the Yellow River in China using combined chemicaland biological tools

Li Wang a,b, Guang-Guo Ying a,*, Feng Chen a, Li-Juan Zhang a, Jian-Liang Zhao a, Hua-Jie Lai a,Zhi-Feng Chen a, Ran Tao a

a State Key Laboratory of Organic Geochemistry, CAS Centre for Pearl River Delta Environmental Pollution and Control Research, Guangzhou Institute of Geochemistry, ChineseAcademy of Sciences, Guangzhou 510640, Chinab South China Institute of Environmental Sciences, Ministry of Environmental Protection, Guangzhou 510655, China

a r t i c l e i n f o

Article history:Received 7 August 2011Received in revised form28 September 2011Accepted 1 October 2011

Keywords:Endocrine disrupting compoundsYeast estrogen screenEstrogenicityYellow RiverRisk assessment

* Corresponding author.E-mail addresses:guangguo.ying@gmail.com, guang-g

0269-7491/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.envpol.2011.10.005

a b s t r a c t

We investigated occurrence of selected compounds (4-t-octylphenol: 4-t-OP; 4-nonylphenols: 4-NP;bisphenol-A: BPA; estrone: E1; 17b-estradiol: E2; triclosan: TCS) and estrogenicity in surface water andsediment of the Yellow River in China by using combined chemical analysis and in vitro yeast screenbioassay. Estrogenic compounds 4-t-OP, 4-NP, BPA, E1, E2 and TCS were measured in the water samples,with their average concentrations of 4.7, 577.9, 46.7, 1.3, ND and 6.8 ng/L, respectively. In sediment, theaverage concentrations of 4-t-OP, 4-NP, BPA and TCS were 35.7, 0.5, 1.7 and 0.7 ng/g while E1 and E2 werenot detected in the sediments of all selected sites. In general, the estrogenic compounds in surface waterand sediment of the Yellow River were at relatively low levels, thus having medium to minimal estro-genic risks in most sites except for the site of east Lanzhou with high estrogenic risks.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Endocrine disrupting chemicals (EDCs) in the environment haveshown great interests in scientific community owing to theirpotential adverse effects in human and wildlife (Colborn et al.,1993; Stone, 1994). One of the most reported biological effects isfish feminization found in various rivers (Angus et al., 2002; Harrieset al., 1997; Jobling et al., 1998; Kristensen et al., 2007; Xie et al.,2010). The observed effects in fish include vitellogenin induction,abnormal development of gonads (Harries et al., 1997; Lye et al.,1997; Jobling et al., 2002) and secondary sexual characteristics(Batty and Lim, 1999; Xie et al., 2010), which have been linked tosome estrogenic compounds in the effluents of sewage treatmentplants (Harries et al., 1997; Jobling et al., 2002; Lye et al., 1997;Thorpe et al., 2003; Xie et al., 2010). These compounds include4-t-octylphenol (4-t-OP), 4-nonylphenols (4-NP), bisphenol-A(BPA), estrone (E1), estradiol (E2) and triclosan (TCS), which havebeen demonstrated to have in vitro and/or in vivo estrogenicactivities (Foran et al., 2000; Ishibahsi et al., 2004; Jobling and

uo.ying@gig.ac.cn (G.-G. Ying).

All rights reserved.

Sumpter, 1993; Jobling et al., 1995; Raut and Angus, 2010;Stasinakis et al., 2008). Therefore, it is essential to monitor theseestrogenic compounds in aquatic environments in order to protectthe ecosystem.

Various chemical and biological tools have been developed andused in the screening of estrogenic compounds in the environment,and the combination of both tools could provide complementaryinformation for contamination assessment (Bicchi et al., 2009;Desbrow et al., 1998; Fu et al., 2007; Labadie and Hill, 2007;Rahman et al., 2009; Streck, 2009). Chemical analysis using gaschromatographyemass spectrometry (GCeMS) or liquid chroma-tographyetandem mass spectrometry (LCeMS/MS) has developedrapidly to provide better systems for identifying EDCs in the envi-ronment (Chen et al., 2010; Kim et al., 2007; Ko et al., 2007; Kolpinet al., 2002; Labadie and Hill, 2007; Micic and Hofmann, 2009; Yinget al., 2009). Several in vitro bioassays (e.g. E-SCREEN, yeastestrogen screen (YES), estrogen responsive chemically activatedluciferase (ER-CALUX) and RTG-2 reporter gene assay) have beendeveloped for determination of the hormonal activity of individualcompounds and environmental samples (Ackermann et al., 2002;Legler et al., 2002; Routledge and Sumpter, 1996; Soto et al.,1995). Bioassays in combination with chemical analysis are valu-able in not only identifying compounds affecting wildlife adversely

L. Wang et al. / Environmental Pollution 165 (2012) 241e249242

but also evaluating the causal links of compounds in the environ-ment by quantifying their proportion of the total estrogenic activityin samples.

The Yellow River, often called “the Mother River of China”, is thesecond longest river in the country. It originates from the Qinghai-Tibet Plateau in the far west of China, flows across nine provinces innorth China, from west to east, with main stream 5464 km long,drainage area 795,000 km2, and average sand content 2.83 kg/m3. Itis a very important water source for about 107 million people ofnorth China, but in recent years it has suffered from low wateryields and water pollution. In some regions of the river basin(e.g. Lanzhou city), national petrochemical plants, mines, metal-lurgical factories and other pollution sources are also located alongthe river. Since effluents from municipal wastewater treatmentplants are the major source of estrogenic compounds in the aquaticenvironment (Ying et al., 2008, 2009), it is crucial to understand thelevels of these estrogenic compounds in the river system and theirpotential adverse effects on aquatic organisms. 4-NP has beenstudied by Xu et al. (2006) andWang et al. (2006), but no publishedinformation has been available on the estrogenic activity and thelevels of other estrogenic compounds in the Yellow River witha high sand content.

The objective of this study was to investigate occurrence ofestrogenic compounds and total estrogenic activity in the YellowRiver by using combined chemical analysis and in vitro bioassay.Selected target estrogenic compounds including 4-t-octylphenol(4-t-OP), 4-nonylphenols (4-NP), bisphenol-A (BPA), estrone (E1),estradiol (E2) and triclosan (TCS) in the surface water and sedimentwere determined using GCeMS, while estrogenic activities ofsurface water and sediment samples were measured using the YES

Fig. 1. Sampling map of the

bioassay. Potential risks to aquatic organisms were assessed basedon the data from GCeMS analysis and YES bioassay.

2. Materials and methods

2.1. Chemicals and materials

The target compounds (4-t-OP, 4-NP, BPA, E1, E2 and TCS) as well as internalstandards 4-n-nonylphenol (4-n-NP), bisphenol-A-d16 (BPA-d16), estrone-2,4,16,16-d4 (E1-d4), and 13C-labeled triclosan (13C12-TCS) were purchased fromSupelco (USA), Dr Ehrenstorfer GmbH (Germany), Cambridge Isotope Laboratories(USA) or Riedel-de-Haën (RDH, Germany). The physiochemical properties of thetarget compounds are given in Table S1 (Supplementary).

The derivatization reagent pentafluorobenzoyl chloride (PFBOCl, purity >99%)was purchased from Aldrich (USA). All reagents of HPLC grade used for sampleprocessing and analysis (methanol, n-hexane, ethyl acetate, toluene, dichloro-methane, triethylamine (TEA) and pyridine) were obtained from Merck Corporation(Shanghai, China). The cartridges used for solid phase extraction (SPE) were OasisHLB cartridges (N-vinylpyrrolidone-m-divinylbenzene copolymer, 500 mg, 6 mL)that were obtained from Waters Corporation (Milford, MA, USA). Glass fiber filters(GF/F, pore size 0.7 mm) were obtained from Whatman (Maidstone, England) andpyrolyzed at 450 �C for 4 h prior to use. Deionized water was prepared with aMilli-Qwater purification system (Millipore, Watford). Stock solutions of chemicals(100 mg/L) were prepared in methanol and stored at �18 �C for later use.

2.2. Sampling sites and sample collection

Sampling sites (15 in total) were selectedmainly in the vicinity with the nationalmonitoring stations from the upper reach to the lower reach of the Yellow River(Fig. 1). Among the 15 sites, 7 sampling sites were located in the upper reach, 3 sitesin the middle reach and 5 sites in the lower reach (Table 1). Lanzhou is the biggestcity in the upper reach with discharge of wastewater into the river, while in themiddle-lower reaches there are cities such as Sanmenxia, Luoyang, Zhengzhou,Xinxiang, Kaifeng and Puyang along the river. With runoff of loess into the YellowRiver, the river bed in the lower reach has been lifted to 2e5 m higher than thesurrounding land, forming the world-famous “aboveground river”. Two sampling

Yellow River in China.

Table 1Site characteristics, and flow and sand content of water in some sampling sites of the Yellow River in dry season and wet season.

Reach Sites Site characteristics May 2008 (wet season) November 2008 (dry season)

Flow (m3/s) Sand (kg/m3) Flow (m3/s) Sand (kg/m3)

Upper Y1 Liujiaxia Reservoir, 80 km away from Lanzhou city 1490 1070Y2 West Lanzhou city with chemical industry, urban areaY3 Central Lanzhou city, urban area 1330 0.17 973 0.10Y4 East Lanzhou city with effluent discharge into the site, urban areaY5 Downstream of Lanzhou city, rural area, light agriculture, very dry landY6 Near Baiyin city surrounded by mining and light agriculture, very dry landY7 Rural area, light agriculture, very dry land

Middle Y8 Tongguan, agriculture and mining 790 2.39 547 2.67Y9 Sanmenxia dam 996 467Y10 Xiaolangdi Reservoir 964 487

Lower Y11 Confluence of several riversY12 Zhengzhou city, urban area 730 0.82 640 0.80Y13 Kaifeng city, urban area 645 485Y14 Rural area, agriculture 786 2.30 518 1.76Y15 Rural area, agriculture 648 2.15 530 1.98

L. Wang et al. / Environmental Pollution 165 (2012) 241e249 243

campaigns were carried out to collect water and sediment samples in thewet season(May 2008) and dry season (November 2008) along the Yellow River. During thesampling, a global position system (GPS) was used to locate the sampling sites.Information of river flow and sand content were collected from some monitoringstations during the sampling periods (Table 1), whereas no relevant data wereavailable for some sites.

Grab water samples were collected in 1 L clean amber glass bottles from 0.5 mbelow the surface. Before sample collection, each bottle was pre-rinsed with riverwater for three times. About 50 mL of methanol and 400 mL of 4 M sulfuric acid wereadded into each bottle to preserve the samples. Then the collected water sampleswere immediately transported in a cooler to the laboratory, and stored at 4 �C beforefiltration and extraction.

The surface sediment samples were collected with a core sampler. The sedi-ments were sieved in the field to remove gravel and plants and stored in 1 L glassbottles. Sodium azide (NaN3, 1 g) was added into each bottle to inhibit microbialgrowth. After returning to the laboratory, the sediments were freeze-dried,homogenized in a glass mortar, passed through a 60 mesh standard sieve andstored at �18 �C until extraction. Total organic carbon (TOC, %) of each sedimentsample was measured with an LECO C230 carbon analyzer (USA) after removal ofcarbonateswith HCl, while sediment particle size distributionwas analyzed by usingthe pipette method (Tables S2 and S3).

2.3. Sample extraction, derivatization and instrumental analysis

The water samples were filtered through pre-baked glass fiber filters (GF/F,Whatman 0.7 mm effective pore size, UK) and were extracted according to themethod described in our previous paper (Zhao et al., 2009). Briefly, exactly 100 mLeach of 1 mg/L of 4-n-NP, BPA-d16, E1-d4 and 13C12-TCSwas added to each sample asthe internal standards. The SPE HLB cartridges were preconditioned with 10 mL ofmethanol and 10 mL of deionized water. Water samples were passed through thecartridges at a flow rate of 5e10 mL/min under vacuum. After dried with air for atleast 1 h, the target compounds were eluted with 7 mL methanol, followed by 5 mLdichloromethane. The extracts were mixed and dried under a gentle nitrogenstream, then redissolved in 1 mL of methanol. Each final extract was then filteredthrough a 0.22 mmmembrane filter into a 2 mL amber glass vial. The vials were keptat �18 �C until GCeMS analysis.

Target compounds in the sediments were extracted by ultrasonic-assistedsolvent extraction. Five grams of the prepared sediments were mixed with theinternal standards (100 ng each of 4-n-NP, BPA-d16, E1-d4 and 13C12-TCS) as well asethyl acetate (10 mL) in a 50 mL of screw-top centrifuge tube. The tube was ultra-sonicated for 15 min and centrifuged at 1370 g for 10 min, and the supernatant wascollected in a 100mL pyriformflask. The sedimentswere extracted for two additionaltimes with 10 mL and 5 mL of ethyl acetate, respectively. The supernatants werecombined and were concentrated to about 1e2 mL on a rotary evaporator (Buchi,Sweden). The extract was further purified with a glass column (6 mm i.d.) loadedwith 1 g of silica gel. The elution was carried out using 6 mL ethyl acetate. The elutewas concentrated to nearly dryness under a gentle nitrogen stream and redissolvedin 1 mL methanol for further treatment. The purified extracts were derivatized withPFBOCl. The target compounds were analyzed using an Agilent 6890N gas chro-matograph (Agilent, USA) equipped with an Agilent 5975B MSD mass spectrometerwith a chemical ionization (CI) source. The detailed derivatization steps andinstrumental analysis procedures were described previously (Zhao et al., 2009).

2.4. Yeast estrogen screen bioassay

The estrogenicity of the samples was determined by using in vitro yeast estrogenscreen (YES) bioassay (Routledge and Sumpter, 1996). Briefly, certain yeast from the

yeast stock stored at �20 �C was added to the growth medium and grown on anorbital shaker for about 24 h until an absorption level (optical density) of 1.0 at620 nm was achieved. The samples were made into a series of 2-fold dilutions forYES assay analysis. The diluted samples (10 mL in ethanol) were seeded into a 96-wellplate. After ethanol was evaporated from the plate, 200 mL of a mixture of red-b-D-galactoside (CPRG) and yeast solution were added to each well using a multi-channel pipette. The solution was prepared by adding the calculated volume ofyeast and 500 mL CPRG (10 mg/mL) to 50 mL minimal medium. The plates wereincubated at 32 �C for 3 days and read at 540 nm for the color development and at620 nm for the blank. The plates were horizontally shaken for 2 min each day duringthe 3 d incubation. The total estrogenic activity in the environmental samples wasmeasured by comparing to the activity of the natural estrogen of E2 and expressed asestradiol equivalent (EEQ). The reporting limit for the YES bioassay was 0.2 ng/L inwater and 0.04 ng/g in sediment.

In addition to the EEQ measured by YES, the theoretical EEQ values for theenvironmental samples were also calculated from chemical analysis of the targetcompounds based on the concept of concentration additivity, which applies since allinvestigated chemicals target the same receptor (Bicchi et al., 2009; Thorpe et al.,2006). Detailed calculation formula and risk assessment methodology for EEQ canbe referred to our previous paper (Zhao et al., 2011). It should be noted thatdiethylstilbestrol (DES) and 17a-ethinylestradiol (EE2) were also included in thechemical analysis, but they were not detected in all samples, thus not reported inthis study.

2.5. Quality assurance and quality control

All data generated from the analysis were subject to strict quality controlprocedures. With each set of samples to be analyzed, a solvent blank, a standard anda procedure blank were run in sequence to check for background contamination,peak identification and quantification. In addition, surrogate standards were addedto all the samples to monitor matrix effects. Recoveries of the six target compoundsranged from 76 to 119% for the spiked water and sediment blank samples (Zhaoet al., 2009). Only trace amount of 4-NP was found in procedural blanks; hencethe background values were not subtracted from the sample measurements. Thelimits of quantification (LOQ) for 4-t-OP, 4-NP, BPA, E1, E2 and TCS in surface waterwere 1, 7, 2, 0.5, 1 and 0.5 ng/L, respectively, while those in sediment were 0.9, 4.9,2.6, 1.1, 3.5 and 0.8 ng/g, respectively.

To avoid contamination during the sampling and sample preparation, samplingbottles and all glassware used in the experiment were cleaned by washing withdetergent, rinsed with deionized water, and burned in a muffle furnace at 450 �C forat least 4 h. All laboratory materials and ware were either made of glass or Teflon toavoid sample contamination.

3. Results

3.1. Estrogenic compounds in surface water and sediment

The six target compounds were detected in surface water fromthe Yellow River in both wet (May) and dry (November) seasons(Fig. 2). 4-t-OP, 4-NP and BPA were detected in all the samplingsites, whereas the detected frequencies of E1 and TCS were 80% and97%, respectively. E2 was only detected in site 4 of November 2008.The concentrations for 4-NP and BPA in the surface water sampleswere in general higher than those for 4-t-OP, E1, E2 and TCS and the

Compounds4-t-OP 4-NP BPA E1 TCS

Con

cent

ratio

n (n

g/L)

10-1

100

101

102

103

104

100% 100% 100% 80% 97%

Surface water

Compounds4-t-OP 4-NP BPA TCS

Con

cent

ratio

n (n

g/g)

0

2

4

50100150200

38% 100% 34% 17%

Sediment

Fig. 2. Concentration ranges of detected target compounds in surface water (ng/L) andsediment (ng/g dw) of the Yellow River, China. The horizontal lines represent 5th, 50th,mean and 95th percentiles, and the boxes represent 25th and 75th percentiles. Medianand mean concentrations are displayed as solid and dashed horizontal lines, respec-tively. Outliers are shown as individual points; and values below the boxes aredetection frequencies (%).

L. Wang et al. / Environmental Pollution 165 (2012) 241e249244

levels of 4-NP were the highest at all sites. The measured concen-trations for the target compounds in the dry season were higherthan in the wet season in most sites (Fig. 3). For example, theconcentrations of 4-NP in surface water fromMay sampling rangedfrom 165.8 to 1187.6 ng/L with a mean of 470.5 ng/L, while those inNovember ranged from 442.3 to 1172.6 ng/L with a mean of685.3 ng/L. The concentrations of E1 in the water from Maysampling ranged from ND to 1.2 ng/L with a mean of 0.5 ng/L, whilethose from November sampling ranged from 0.7 to 15.6 ng/L witha mean of 2.2 ng/L.

These target compounds varied widely spatially, with highconcentrations being found more frequently at sites 4, 8 and 11(Tables S4 and S5). From the data for the November sampling, thehighest concentrations for four out of the six compounds in surfacewater were found in site 4, which is located at the downstream ofeffluent discharge point of Lanzhou sewage treatment plant.

The six estrogenic compounds were detected in the sedimentsof the Yellow River, except for E1 and E2 below their LOQ or notdetected in any samples from the river (Fig. 2). 4-NP was the mostdetected target compound, ranging between 25.5 and 46.5 ng/gwith a mean of 33.5 ng/g in May and between 16.6 and 203.8 ng/gwith a mean of 38.0 ng/g in November. Seasonally, 4-t-OP and 4-NPin the sediment had higher detection frequencies in the dry seasonthan in the wet season (Fig. 4). Spatially, the highest concentrationsfor the detected four compounds were observed in site 4 (Fig. 4;Tables S6 and S7).

3.2. Estrogenicity in surface water and sediment

The estrogenicity in surface water measured by the YES assay inNovember (dry season) and May (wet season) is shown in Fig. 5. Inthe wet season, only site 8 was found to have the EEQ value abovethe reporting limit (0.2 ng/L), with the concentration of 0.21 ng/L. Inthe dry season, eight sites (Y3e9 and Y11) out of 15 sites werefound to have the EEQ above the reporting limit, with the highestconcentration up to 9.44 ng/L at site 4 (Table S8).

Estrogenic activities in the sediments from the Yellow Riverwere also observed in both seasons (Fig. 5). Only seven sites in wetseason and three sites in dry season out of the 15 sites were foundto have the EEQ above the reporting limit (0.04 ng/g). The highestEEQ value in the wet season was found at site 7 with the concen-tration of 1.29 ng/g, while that in the dry seasonwas found at site 4with the concentration of 0.45 ng/g (Table S8).

4. Discussion

4.1. Distribution of estrogenic compounds

The occurrence of six estrogenic compounds was investigated insurface water of the Yellow River with concentrations ranging frombelow detection to several thousand ng/L. 4-NP, a degradationproduct of the surfactant nonylphenol polyethoxylates (Ying et al.,2002), was detected in all samples of the Yellow River at theconcentrations ranging from 165.8 to 1187.6 ng/L. The concentra-tion range for 4-NP found in the present study is consistent withprevious results in the Yellow River reported by Xu et al. (2006) andWang et al. (2006) from 34.2 to 599 ng/L and 50 to 170 ng/L,respectively. 4-t-OP and BPAwere also detected at all sampling sites(Table 2) and have been widely detected in aquatic environments,usually at levels of several to hundreds of nanograms per liter (Boydet al., 2004; Furuichi et al., 2004; Kuch and Ballschmiter, 2001).Both levels and detection frequencies of 4-t-OP and BPA in thewater of the Yellow River appear lower than those in the Pearl River(Zhao et al., 2009), Hai River (Jin et al., 2004) of China, the TamaRiver of Japan (Furuichi et al., 2004), but similar to those in the OuseRiver of UK (Zhang et al., 2006) and Venice lagoon of Italy (Pojanaet al., 2007) (Table 2). TCS is a broad-spectrum antimicrobial andpreservative agent that is widely used in personal care products(Stasinakis et al., 2008) and it had a frequency of detection of 97% inthe Yellow River. This indicates contamination of domestic sewagein the Yellow River. The concentrations of TCS in the Yellow Riverare higher than those of the river water in Korea (Table 2). However,much higher concentrations of TCS in surface water were reportedby Kolpin et al. (2002) with the maximum concentration up to2300 ng/L in US streams.

Estrone (E1) was the most frequently detected estrogen (80%)with a maximum concentration of 1.2 ng/L in May and 15.6 ng/Lin November (Table 2). The concentrations of E1 in the Yellowriver water are lower than those in the Pearl River (Zhao et al.,2009), Dan-Shui River of China (Chen et al., 2007), Tama Riverof Japan (Furuichi et al., 2004) and streams of USA (Kolpin et al.,2002), but higher than those in the lakes and rivers of Korea (Kimet al., 2007) and the Venice lagoon of Italy (Pojana et al., 2007),and comparable to those in surface water samples of Australia(Hohenblum et al., 2004; Ying et al., 2009) (Table 2). Estradiol(E2) was only detected at 2.3 ng/L in site 4 of the Yellow River.The site is near the effluent outfall of Lanzhou sewage treatmentplant. The infrequent detection is due to the easy degradation ofE2 in the environment (Ternes et al., 1999; Ying and Kookana,2005).

The concentration ranges for the estrogenic compounds in thesediments of the Yellow River are mostly lower than those

Y1 Y2 Y3 Y4 Y5 Y6 Y7 Y8 Y9 Y10

Y11

Y12

Y13

Y14

Y15

Con

cent

ratio

n (n

g/L)

02468

10121416

Wet season Dry season

4-t-OP

Y1 Y2 Y3 Y4 Y5 Y6 Y7 Y8 Y9 Y10

Y11

Y12

Y13

Y14

Y15

Con

cent

ratio

n (n

g/L)

0200400600800

1000120014001600

4-NP

Y1 Y2 Y3 Y4 Y5 Y6 Y7 Y8 Y9 Y10

Y11

Y12

Y13

Y14

Y15

Con

cent

ratio

n (n

g/L)

020406080

100120140160180200

BPA

Y1 Y2 Y3 Y4 Y5 Y6 Y7 Y8 Y9 Y10

Y11

Y12

Y13

Y14

Y15

Con

cent

ratio

n (n

g/L)

0123

14

16

18 E1

Y1 Y2 Y3 Y4 Y5 Y6 Y7 Y8 Y9 Y10

Y11

Y12

Y13

Y14

Y15

Con

cent

ratio

n (n

g/L)

0.0

.5

1.0

1.5

2.0

2.5E2

Y1

Y2

Y3

Y4

Y5

Y6

Y7

Y8

Y9

Y10

Y11

Y12

Y13

Y14

Y15

Con

cent

ratio

n (n

g/L)

05

101520406080 TCS

Fig. 3. Distribution of target compounds in surface water of the Yellow River in the wet season and dry season. The error bars represent the standard deviation of themeasurements.

L. Wang et al. / Environmental Pollution 165 (2012) 241e249 245

previously reported in the literature (Table 2). The estrogens (E1and E2) were not detected in all the samples, similar to those in thePearl River (Zhao et al., 2009); however, they were detected in theVenice lagoon of Italy (Pojana et al., 2007) with their maximum

Y1 Y2 Y3 Y4 Y5 Y6 Y7 Y8 Y9 Y10

Y11

Y12

Y13

Y14

Y15

Con

cent

ratio

n (n

g/g)

0.0

.5

1.0

1.5

2.0

2.5

3.0Wet season Dry season

4-t-OP

Y1 Y2 Y3 Y4 Y5 Y6 Y7 Y8 Y9 Y10

Y11

Y12

Y13

Y14

Y15

Con

cent

ratio

n (n

g/g)

0

2

4

6

8

10BPA

Fig. 4. Distribution of target compounds in sediment of the Yellow River in the wet season

concentration of 7 and 11.2 ng/g, respectively. The Yellow River hashigh sand contents and low TOC contents in its surface water andsediment (Tables S2 and S3), which results in low detectionfrequencies and low concentrations of these target compounds.

Y1 Y2 Y3 Y4 Y5 Y6 Y7 Y8 Y9 Y10

Y11

Y12

Y13

Y14

Y15

Con

cent

ratio

n (n

g/g)

0

20

40

180

200

220

2404-NP

Y1 Y2 Y3 Y4 Y5 Y6 Y7 Y8 Y9 Y10

Y11

Y12

Y13

Y14

Y15

Con

cent

ratio

n (n

g/g)

0

1

2

5

10

15TCS

and dry season. The error bars represent the standard deviation of the measurements.

Table

2Com

parison

sof

concentrationrange

sof

target

compou

ndsin

water

andsedim

entbe

twee

ntheYe

llow

River

andother

sitesin

theworld.

Com

pou

nds

Asia

Europe

Australia

America

TheYellow

River

ThePe

arlRiver

TheHai

River

Dan

-ShuiRiver

Korea

Japan

UK

Italy

Australia

USA

Water

4-t-OP

2.4e

14.5

(4.3)a

1.0e

2470

(17.4)

18.0e20

.2(19.3)

ND

NDe81

.9<0.3e

65NDe94

4-NP

165.8e

1187

.6(534

)28

.1e88

90(96)

106e

296(191

)NDe24

4.8

NDe10

80<0.2e

8<0.5e

211(29)

NDe20

58NDe40

,000

(800

)BPA

12.5e17

1.5(39.5)

2.2e

1030

(132

)19

.1e10

6(34.8)

NDe39

.4NDe60

0<0.1e

48<1.0e

145(7.6)

NDe60

0NDe12

,000

(140

)E1

NDbe15

.61.3(0.9)

NDe75

(2.8)

22.4e66

.2(31.1)

1.7e

5.0

NDe85

.6<0.3e

41<1.2e

1.0(<

1.2)

NDe20

.9NDe11

2(27)

E2NDe2.3(<

LOQ)c

NDe7.5(1.2)

1.4e

33.9

(14.5)

ND

NDe12

.3<LO

Qe16

<1.0e

36(<

1.0)

NDe3.77

NDe20

0(160

)TC

SNDe49

.9(4.2)

0.6e

347(14.7)

ND

NDe23

00(140

)Se

dim

ent

4-t-OP

NDe2.6(<

LOQ)

<LO

Qe97

93.97

e17

93e

670

<LO

Qe18

00NDe12

.5(N

D)

4-NP

16.6e20

3.8(27.8)

11.4e28

,830

10.4e50

54.1

30e13

,000

<LO

Qe72

,000

47e19

2(82)

NDe22

.9(N

D)

BPA

NDe7.7(<

LOQ)

<LO

Qe29

62.7e

50.3

<LO

Qe56

.1<2.0e

118(32)

NDe5.0(N

D)

E1ND

(ND)

NDe38

.0<LO

Qe7

E2ND

(ND)

NDe4.1

<LO

Qe11

.2TC

SNDe14

.0(N

D)

Referen

ces

This

study

Chen

etal.,20

06;

Penget

al.,20

06;

Zhao

etal.,

2009

,201

1

Jinet

al.,20

04Chen

etal.,20

07Khim

etal.,19

99;

Koet

al.,20

07;

Kim

etal.,20

07;

Liet

al.,20

04

Furuichie

tal.,20

04;

Hoh

enblum

etal.,20

04;

Isob

eet

al.,20

01

Hibbe

rdet

al.,20

09;

Liuet

al.,20

04;

Petrov

icet

al.,20

02;

Zhan

get

al.,20

06

Pojana

etal.,20

07Hoh

enblum

etal.,20

04;

Ying

etal.,20

09

Kolpin

etal.,20

02;

Stuartet

al.,20

05

aCon

centrationrange

:minim

um

tomax

imum

(med

ian).

bND:not

detected.

c<LO

Q:be

low

thelim

itof

quan

titation

.

Sampling sites

Y1 Y2 Y3 Y4 Y5 Y6 Y7 Y8 Y9 Y10

Y11

Y12

Y13

Y14

Y15

Det

erm

ined

EEQ

(ng/

L)

0.0.2.3.4

4.0

8.0

12.0Wet season

Dry season

Surface water

Sampling sites

Y1 Y2 Y3 Y4 Y5 Y6 Y7 Y8 Y9 Y10

Y11

Y12

Y13

Y14

Y15

Det

erm

ined

EEQ

(ng/

g)

0.0

.3

.6

.9

1.2

1.5

1.8Wet season

Dry seasonSediment

Fig. 5. Distribution of measured EEQs in surface water and sediment of the YellowRiver in the dry season and wet season. EEQ: Estradiol equivalent.

L. Wang et al. / Environmental Pollution 165 (2012) 241e249246

Seasonal and spatial variations in concentrations of the sixestrogenic compounds were observed in surface water and sedi-ment of the Yellow River. The concentrations of 4-t-OP, 4-NP, BPA,E1, E2 and TCS in the dry season (November, winter) were generallyhigher than those in thewet season (May, summer). Ko et al. (2007)also measured higher concentrations in river waters of Korea inwinter than in summer. This is mainly due to the effects caused bydilution and temperature change. In the sampling sites, water flowis higher inMay than in November (Table 1). For example, thewaterflows of central Lanzhou (site 3) were 1330 and 973 m3/s in Mayand November, respectively. High flow leads to dilution ofcontaminants and movement of settled sediment. In addition,temperature may also be a factor for the higher concentrations inthe dry season. In November, the temperature in the region coulddrop tominus degrees, which can reduce themicrobial degradationof these contaminants in the river.

The concentrations for the six target compounds variedspatially, with the high concentrations inwater and sediment beingfound more frequently at sites 4, 8, 11 and 4e6, respectively. Thehighest concentrations for most target compounds in surface waterand sediment were detected in east Lanzhou (site 4). The site islocated not far from the effluent outfall of Lanzhou sewage treat-ment plant. Niu et al. (2006) also found the highest concentrationsof polycyclic aromatic hydrocarbons (PAHs) in the site. Lanzhou asthe capital of Gansu province is an industrial city with variousindustries such as oil refineries, petrochemical plants and rubberfactories. Domestic and industrial wastewaters are discharged intothe river section, which affects the water quality of the Yellow River(Wei, 1998).

Distribution of contaminants in the sediments of a river is alsorelated to sediment properties such as organic carbon, and particlesize distribution (Baker et al., 1991; Lai et al., 2000). Weak corre-lations of the contaminant concentrations and TOC were found forthe four compounds 4-NP, 4-t-OP, BPA and TCS, but no correlationswere found between the contaminant concentrations and claycontent (Fig. 6 and Fig. S1). Owing to high river flows and sandcontents in the Yellow River, fine sediment is easily stirred andcarried away by the river water. Due to sandy nature of the sedi-ments in the Yellow River, adsorption of these compounds was very

TOC (%)

0.0 .2 .4 .6

Co

nc

en

tra

tio

n (n

g/g

)

0.0

.5

1.0

1.5

2.0

2.5

3.0

4-t-OP y=1.5x+0.2

R2

=0.1413

F=5 p=0.0406

TOC (%)

0.0 .2 .4 .6

Co

nc

en

tra

tio

n (n

g/g

)

0

50

100

150

200

250

4-NP y=117.3x+14.8

R2

=0.3507

F=15 p=0.0006

TOC (%)

0.0 .2 .4 .6

Co

nc

en

tra

tio

n (n

g/g

)

0

2

4

6

8

10

BPA y=5.5x+0.7

R2

=0.1338

F=4 p=0.0468

TOC (%)

0.0 .2 .4 .6

Co

nc

en

tra

tio

n (n

g/g

)02468

10121416

TCS y=9.6x-1.0

R2

=0.3992

F=19 p=0.0002

Fig. 6. Correlation of concentrations of target compounds with total organic carbon contents (TOC, %) of sediment in the Yellow River.

Calculated EEQ (ng/L)

0 2 4 6 8 10 12

Determ

in

ed

E

EQ

(n

g/L

)

0

2

4

6

8

10

12

Surface water

y=0.3x-0.1

R2=0.5164

F=29 p<0.0001

Calculated EEQ (ng/g)

0.000 .005 .010 .015 .020 .025

Determ

in

ed

E

EQ

(n

g/g

)

0.0

.2

.4

.6

.8

1.0

1.2

1.4

y=18.8x+0.1

R2

=0.0615

F=2 p=0.1866

Sediment

Fig. 7. Relationship of measured EEQs and calculated EEQs in surface water andsediment of the Yellow River. EEQ: Estradiol equivalent.

L. Wang et al. / Environmental Pollution 165 (2012) 241e249 247

weak based on the calculated pseudo-partitioning coefficients:0e598 L/kg, 14e287 L/kg, 0e192 L/kg and 0e383 L/kg for 4-t-OP, 4-NP, BPA and TCS. These calculated sorption coefficients are muchlower than those reported values (Table S1), suggesting pooradsorption of these compounds on sandy sediments of the YellowRiver.

4.2. Comparison of estrogenicity by YES bioassay and chemicalanalysis

The theoretical estrogenicity calculated based on chemicalanalysis had a good linear relationship with the total estrogenicitymeasured by the YES bioassay for surface water in the Yellow River(Fig. 7). The theoretical estrogenicity was calculated as thesummation of individual estrogenicity estimated from eachchemical concentration monitored by GCeMS (Bicchi et al., 2009;Zhao et al., 2011).

For most water samples, the EEQ values determined by the YESassay were lower than those calculated from chemical analysis(Table S8). This can be explained by the presence of unknownantagonists in the water samples (Tanaka et al., 2001; Witters et al.,2001). Somewater extracts exerted toxicity to the yeast cells duringthe bioassay, which affected development of color and furthermeasurement of EEQ. Similar toxic effects on yeast cells have beenreported previously in wastewater (Tanaka et al., 2001) and surfacewater (Witters et al., 2001).

For most sediment samples, the EEQ values determined by theYES bioassay were higher than those calculated from chemicalanalysis (Table S8). This suggests that some unknown chemicalsmight contribute to the estrogenicity of the samples (Sarmah et al.,2006; Soto et al., 2004; Viganò et al., 2008).

The present study demonstrates that estrogenic activity ina sample may not be fully explained by chemical analysis of theselected target compounds, and an estrogenic screening bioassaysuch as the YES assay could provide the overall estrogenicity of

a sample. Therefore, theEEQmeasuredby theYES in thepresent studycould be used in the further risk assessment for the Yellow River.

4.3. Risk assessment

The presence of estrogenic compounds and their estrogenicactivities might affect aquatic organisms such as fish in rivers by

L. Wang et al. / Environmental Pollution 165 (2012) 241e249248

disrupting the normal hormonal functions of these organisms(Jobling et al., 1998; Sumpter, 1998; Tyler and Routledge, 1998).According to the no-observed-effect-concentrations of E2 in USEPAECOTOX database, we calculated the predicated no-effect concen-trations (PNEC) of 1.5 ng/L (Zhao et al., 2011). Risk quotients (RQs)were obtained from the ratio of the measured EEQs and PNEC. Onlythe RQ value of site 4 in the dry season was more than 1 (Fig. 5),indicating it could pose a high risk to aquatic organisms. The RQvalue of sites 3, 5e9 and 11 in dry season and site 8 in wet seasonranged between 0.1 and 1, indicating it could pose a medium risk toaquatic organisms. The present study showed that Lanzhou sectionhad the most serious estrogenic pollution problem. However, thisfirst-tier risk assessment for the Yellow river was based on thein vitro bioassay data, the predicated estrogenic risks may/may notbe reflected in the aquatic organisms in the Yellow River. Therefore,a further biological exposure study is needed to establish the realconsequences of estrogenic activity in the Yellow River.

5. Conclusions

The occurrence of six estrogenic compounds (4-t-OP, 4-NP, BPA,TCS, E1 and E2) and total estrogenicity in the Yellow River wasassessed by using the combined chemical and biological analyses.Surfactant degradation product 4-NP was the most frequentlydetected compound inwater and sediment, while two estrogens E1and E2 were not detected in the sediments from all sampling sites.The measured concentrations for the target compounds in the dryseason were higher than in the wet season in most sites. Theconcentrations of these compounds in the sediments of the YellowRiver were relatively lowcomparingwith the reported data in someother regions, mainly due to poor adsorption of these compoundson the sandy sediments. The highest concentrations for the targetcompounds were found more frequently at the site of east Lanzhouwith sewage effluent discharge. The results from chemical analysisand in vitro bioassay showed highest estrogenic risks in Lanzhousection of the river in the dry season. Proper measures should beadopted to reduce the discharge of sewage effluents containingestrogenic compounds into the river system in order to protectaquatic organisms in the river.

Acknowledgments

Wewish to acknowledge the financial support from the NationalNatural Science Foundation of China (NSFC 40821003, 20977092and 40688001), Guangdong Provincial Natural Science Foundation(8251064004000001), the Earmarked Fund from the State KeyLaboratory of Organic Geochemistry (SKLOG2009A02) and theChinese Academy of Sciences (KZCX2-EW-108). This is a Contribu-tion No. 1395 from GIG CAS.

Appendix. Supplementary material

Supplementary material associated with this article can befound, in the online version, at doi:10.1016/j.envpol.2011.10.005.

References

Ackermann, G.E., Brombacher, E., Fent, K., 2002. Development of a fish reportergene system for the assessment of estrogenic compounds and sewage treat-ment plant effluents. Environmental Toxicology and Chemistry 21, 1864e1875.

Angus, R.A., Weaver, S.A., Grizzle, J.M., Watson, R.D., 2002. Reproductive charac-teristics of male mosquitofish (Gambusia affinis) inhabiting a small southeasternUS river receiving treated domestic sewage effluent. Environmental Toxicologyand Chemistry 21, 1404e1409.

Baker, J.E., Eisenreich, Eadie, S.J., 1991. Sediment trap fluxes and benthic recycling oforganic carbon, polycyclic aromatic hydrocarbons, and polychlorobiphenylcongeners in Lake Superior. Environmental Science and Technology 25, 500e509.

Batty, J., Lim, R.P., 1999. Morphological and reproductive characteristics of malemosquitofish (Gambusia affinis holbrooki) inhabiting sewage contaminatedwaters in New South Wales, Australia. Archives of Environmental Contamina-tion and Toxicology 36, 301e307.

Bicchi, C., Schilir, T., Pignata, C., Fea, E., Cordero, C., Canale, F., Gilli, G., 2009. Analysisof environmental endocrine disrupting chemicals using the E-screen methodand stir bar sorptive extraction in wastewater treatment plant effluents. Scienceof the Total Environment 407, 1842e1851.

Boyd, G.R., Palmeri, J.M., Zhang, S., Grimm, D.A., 2004. Pharmaceuticals andpersonal care products (PPCPs) and endocrine disrupting chemicals (EDCs) instormwater canals and Bayou St. John in New Orleans, Louisiana, USA. Scienceof the Total Environment 333, 137e148.

Chen, B., Duan, J.C., Mai, B.X., Luo, X.J., Yang, Q.S., Sheng, G.Y., Fu, J.M., 2006.Distribution of alkylphenols in the Pearl River Delta and adjacent northernSouth China Sea, China. Chemosphere 63, 652e661.

Chen, C.Y., Wen, T.Y., Wang, G.S., Cheng, H.W., Lin, Y.H., Lien, G.W., 2007. Deter-mining estrogenic steroids in Taipei waters and removal in drinking watertreatment using high-flow solid-phase extraction and liquid chromatography/tandem mass spectrometry. Science of the Total Environment 378, 352e365.

Chen, F., Ying, G.G., Yang, J.F., Zhao, J.L., Wang, L., 2010. Rapid resolution liquidchromatography-tandem mass spectrometry method for the determination ofendocrine disrupting chemicals (EDCs), pharmaceuticals and personal careproducts (PPCPs) in wastewater irrigated soils. Journal of EnvironmentalScience and Health Part B 45, 682e693.

Colborn, T., vom Saal, F.S., Soto, A.M., 1993. Developmental effects of endocrine-disrupting chemicals in wildlife and humans. Environmental Health Perspec-tives 101, 378e384.

Desbrow, C., Routledge, E.J., Brighty, G.C., Sumpter, J.P., Waldock, M., 1998. Identi-fication of estrogenic chemicals in STW effluent. 1. Chemical fractionation andin vitro biological screening. Environmental Science and Technology 32,1549e1558.

Foran, C.M., Bennett, E.R., Benson, W.H., 2000. Developmental evaluation ofa potential non-steroidal estrogen: triclosan. Marine Environmental Research50, 153e156.

Fu, K.Y., Chen, C.Y., Chang, W.M., 2007. Application of a yeast estrogen screen innon-biomarker species Varicorhinus barbatulus fish with two estrogen receptorsubtypes to assess xenoestrogens. Toxicology in Vitro 21, 604e612.

Furuichi, T., Kannan, K., Giesy, J.P., Masunaga, S., 2004. Contribution of knownendocrine disrupting substances to the estrogenic activity in Tama River watersamples from Japan using instrumental analysis and in vitro reporter geneassay. Water Research 38, 4491e4501.

Harries, J.E., Sheahan, D.A., Jobling, S., Matthiessen, P., Neall, M., Sumpter, J.P.,Taylor, T., Zaman, N., 1997. Estrogenic activity in five United Kingdom riversdetected by measurement of vitellogenesis in caged male trout. EnvironmentalToxicology and Chemistry 16, 534e542.

Hibberd, A., Maskaoui, K., Zhang, Z., Zhou, J.L., 2009. An improved method for thesimultaneous analysis of phenolic and steroidal estrogens in water and sedi-ment. Talanta 77, 1315e1321.

Hohenblum, P., Gans, O., Moche, W., Scharf, S., Lorbeer, G., 2004. Monitoring ofselected estrogenic hormones and industrial chemicals in groundwaters andsurface waters in Austria. Science of the Total Environment 333, 185e193.

Ishibahsi, H., Matsumura, N., Hirano, M., Matsuoka, M., Shiratsuchi, H., Ishibashi, Y.,Takao, Y., Arizono, K., 2004. Effects of triclosan on the early life stages andreproduction of medaka Oryzias latipes and induction of hepatic vitellogenin.Aquatic Toxicology 67, 167e179.

Isobe, T., Nishiyama, H., Nakashima, A., Takada, H., 2001. Distribution and behaviorof nonylphenol, octylphenol, and nonylphenol monoethoxylate in Tokyometropolitan area: their association with aquatic particles and sedimentarydistributions. Environmental Science and Technology 35, 1041e1049.

Jin, X., Jiang, G., Huang, G., Liu, J., Zhou, Q., 2004. Determination of 4-tert-octyl-phenol, 4-nonylphenol and bisphenol A in surface waters from the Hai River inTianjin by gas chromatography-mass spectrometry with selected ion moni-toring. Chemosphere 56, 1113e1119.

Jobling, S., Nolan, M., Tyler, C.R., Brighty, G., Sumpter, J.P., 1998. Widespread sexualdisruption in wild fish. Environmental Science and Technology 32, 2498e2506.

Jobling, S., Sumpter, J.P., 1993. Detergent components in sewage effluent are weaklyestrogenic to fish e an in-vitro study using rainbow-trout (Oncorhynchus-mykiss) hepatocytes. Aquatic Toxicology 27, 361e372.

Jobling, S., Reynolds, T., White, R., Parker, M.G., Sumpter, J.P., 1995. A variety ofenvironmentally persistent chemicals, including some phthalate plasticizers,are weakly estrogenic. Environmental Health Perspectives 103, 582e587.

Jobling, S., Beresford, N., Nolan, M., Rodgers-Gray, T., Brighty, G.C., Sumpter, J.P.,Tyler, C.R., 2002. Altered sexual maturation and gamete production in wildroach (Rutilis rutilis) living in rivers that receive treated sewage effluents.Biology of Reproduction 66, 272e281.

Khim, J.S., Kannan, K., Villeneuve, D.L., Koh, C.H., Giesy, J.P., 1999. Characterizationand distribution of trace organic contaminants in sediment from Masan bay,Korea. 1. Instrumental analysis. Environmental Science and Technology 33,4199e4205.

Kim, S.D., Cho, J., Kim, I.S., Vanderford, B.J., Snyder, S.A., 2007. Occurrence andremoval of pharmaceuticals and endocrine disruptors in South Korean surface,drinking, and waste waters. Water Research 41, 1013e1021.

Ko, E.J., Kim, K.W., Kang, S.Y., Kim, S.D., Bang, S.B., Hamm, S.Y., Kim, D.W., 2007.Monitoring of environmental phenolic endocrine disrupting compounds intreatment effluents and river waters, Korea. Talanta 73, 674e683.

L. Wang et al. / Environmental Pollution 165 (2012) 241e249 249

Kolpin, D.W., Furlong, E.T., Meyer, M.T., Thurman, E.M., Zaugg, S.D., Barber, L.B.,Buxton, H.T., 2002. Pharmaceuticals, hormones, and other organic wastewatercontaminants in U.S. streams, 1999e2000: a national reconnaissance. Envi-ronmental Science and Technology 36, 1202e1211.

Kristensen, T., Edwards, T.M., Kohno, S., Baatrup, E., Guillette, L.J.J., 2007. Fecundity,17b-estradiol concentrations and expression of vitellogenin and estrogenreceptor genes throughout the ovarian cycle in female eastern mosquitofishfrom three lakes in Florida. Aquatic Toxicology 81, 245e255.

Kuch, H.M., Ballschmiter, K., 2001. Determination of endocrine-disrupting phenoliccompounds and estrogens in surface and drinking water by HRGC-(NCI)-MS inthe picogram per liter range. Environmental Science and Technology 35,3201e3206.

Labadie, P., Hill, E.M., 2007. Analysis of estrogens in river sediments by liquidchromatography-electrospray ionisation mass spectrometry: comparison oftandem mass spectrometry and time-of-flight mass spectrometry. Journal ofChromatography A 1141, 174e181.

Lai, K.M., Johnson, K.L., Scrimshaw, M.D., Lester, J.N., 2000. Binding of waterbornesteroid estrogens to solid phases in river and estuarine systems. EnvironmentalScience and Technology 34, 3890e3894.

Legler, J., Dennekamp, M., Vethaak, A.D., Brouwer, A., Koeman, J.H., van der Burg, B.,Murk, A.J., 2002. Detection of estrogenic activity in sediment-associatedcompounds using in vitro reporter gene assays. The Science of the Total Envi-ronment 293, 69e83.

Li, Z., Li, D., Oh, J.R., Je, J.G., 2004. Seasonal and spatial distribution of nonylphenol inShihwa Lake, Korea. Chemosphere 56, 611e618.

Liu, R., Zhou, J.L., Wilding, A., 2004. Simultaneous determination of endocrinedisrupting phenolic compounds and steroids in water by solid-phase extrac-tion-gas chromatography-mass spectrometry. Journal of Chromatography A1022, 179e189.

Lye, C.M., Frid, C.L.J., Gill, M.E., McCormick, D., 1997. Abnormalitiesin the repro-ductive health of flounder Platichthys flesus exposed to effluent from a sewagetreatment works. Marine Pollution Bulletin 34, 34e41.

Micic, V., Hofmann, T., 2009. Occurrence and behaviour of selected hydrophobicalkylphenolic compounds in the Danube River. Environmental Pollution 157,2759e2768.

Niu, J.P., Liu, Y.P., Ruan, Y., Ding, G.W., 2006. The environmental hormone pollutionin Lanzhou of the Yellow River. Journal of Environment and Health 23,527e529.

Peng, X.Z., Wang, Z.D., Yang, C., Chen, F.R., Mai, B.X., 2006. Simultaneous determi-nation of endocrine-disrupting phenols and steroid estrogens in sediment bygas chromatographyemass spectrometry. Journal of Chromatography A 1116,51e56.

Petrovic, M., Eljarrat, E., López de Alda, M.J., Barceló, D., 2002. Recent advances inthe mass spectrometric analysis related to endocrine disrupting compounds inaquatic environmental samples. Journal of Chromatography A 974, 23e51.

Pojana, G., Gomiero, A., Jonkers, N., Marcomini, A., 2007. Natural and syntheticendocrine disrupting compounds (EDCs) in water, sediment and biota ofa coastal lagoon. Environment International 33, 929e936.

Rahman, M.F., Yanful, E.K., Jasim, S.Y., 2009. Occurrences of endocrine disruptingcompounds and pharmaceuticals in the aquatic environment and their removalfrom drinking water: challenges in the context of the developing world.Desalination 248, 578e585.

Raut, S.A., Angus, R.A., 2010. Triclosan has endocrine disrupting effects in malewestern mosquitofish, Gambusia affinis. Environmental Toxicology and Chem-istry 29, 1287e1291.

Routledge, E.J., Sumpter, J.P., 1996. Estrogenic activity of surfactants and some oftheir degradation products assessed using a recombinant yeast screen. Envi-ronmental Toxicology and Chemistry 15, 241e248.

Sarmah, A.K., Northcott, G.L., Leusch, F.D.L., Tremblay, L.A., 2006. A survey ofendocrine disrupting chemicals (EDCs) in municipal sewage and animal wasteeffluents in the Waikato region of New Zealand. Science of the Total Environ-ment 355, 135e144.

Soto, A.M., Sonnenschein, C., Chung, K.L., Fernandez, M.F., Olea, N., Serrano, F.O.,1995. The E-screen assay as a tool to identify estrogens: an update on estrogenicenvironmental pollutants. Environmental Health Perspectives 103, 113e122.

Soto, A.M., Calabro, J., Prechtl, N.V., Yau, A.Y., Orlando, E.F., Daxenberger, A., Kolok, A.S.,Guillette, L.J., le Bizec, B., Lange, I.G., Sonnenschein, C., 2004. Androgenic andestrogenic activity in water bodies receiving cattle feedlot effluent in EasternNebraska, USA. Environmental Health Perspectives 112, 346e352.

Stasinakis, A.S., Gatidou, G., Mamais, D., Thomaidis, N.S., Lekkas, T.D., 2008.Occurrence and fate of endocrine disrupters in Greek sewage treatment plants.Water Research 42, 1796e1804.

Stone, R., 1994. Environmental estrogens stir debate. Science 265, 308e310.Streck, G., 2009. Chemical and biological analysis of estrogenic, progestagenic and

androgenic steroids in the environment. Trends in Analytical Chemistry 28,635e652.

Stuart, J.D., Capulong, C.P., Launer, K.D., Pan, X., 2005. Analyses of phenolic endo-crine disrupting chemicals in marine samples by both gas and liquid chroma-tographyemass spectrometry. Journal of Chromatography A 1079, 136e145.

Sumpter, J.P., 1998. Xenoendocrine disrupters-environmental impacts. ToxicologyLetters 102-103, 337e342.

Tanaka, H., Yakou, Y., Takahashi, A., Higashitani, T., Komori, K., 2001. Comparisonbetween estrogenicities estimated from DNA recombinant yeast assay and fromchemical analyses of endocrine disruptors during sewage treatment. WaterScience and Technology 43, 125e132.

Ternes, T.A., Kreckel, P., Mueller, J., 1999. Behaviour and occurrence of estrogens inmunicipal sewage treatment plants-II. Aerobic batch experiments with acti-vated sludge. Science of the Total Environment 225, 91e99.

Thorpe, K.L., Cummings, R.I., Hutchinson, T.H., Scholze, M., Brighty, G., Sumpter, J.P.,Tyler, C.R., 2003. Relative potencies and combination effects of steroidalestrogens in fish. Environmental Science and Technology 37, 1142e1149.

Thorpe, K.L., Gross-Sorokin, M., Johnson, I., Brighty, G., Tyler, C.R., 2006. Anassessment of the model of concentration addition for predicting the estrogenicactivity of chemical mixtures in wastewater treatment works effluents. Envi-ronmental Health Perspectives 114, 90e97.

Tyler, C.R., Routledge, E.J., 1998. Oestrogenic effects in fish in English rivers withevidence of their causation. Pure and Applied Chemistry 70, 1795e1804.

Viganò, L., Benfenati, E., Cauwenberge, A.V., Eidem, J.K., Erratico, C., Goksøyr, A.,Kloas, W., Maggioni, S., Mandich, A., Urbatzka, R., 2008. Estrogenicity profileand estrogenic compounds determined in river sediments by chemical analysis,ELISA and yeast assays. Chemosphere 73, 1078e1089.

Wang, L., Wu, Y., Sun, H., Xu, J., Dai, S., 2006. Distribution and dissipation pathwaysof nonylphenol polyethoxylates in the Yellow River: site investigation and lab-scale studies. Environment International 32, 907e914.

Wei, J., 1998. Water pollution and control of Lanzhou. Development and Research 5,33e36.

Witters, H.E., Vangenechten, C., Berckmans, P., 2001. Detection of estrogenic activityin flemish surface waters using an in vitro recombinant assay with yeast cells.Water Science and Technology 43, 117e123.

Xie, Y.P., Fang, Z.Q., Hou, L.P., Ying, G.G., 2010. Altered development and repro-duction in western mosquitofish (Gambusia affinis) found in the Hanxi River,southern China. Environmental Toxicology and Chemistry 29, 2607e2615.

Xu, J., Wang, P., Guo, W., Dong, J., Wang, L., Dai, S., 2006. Seasonal and spatialdistribution of nonylphenol in Lanzhou reach of Yellow River in China. Che-mosphere 65, 1445e1451.

Ying, G.G., Kookana, R.S., 2005. Sorption and degradation of estrogen-like-endocrine disrupting chemicals in soil. Environmental Toxicology and Chem-istry 24, 2640e2645.

Ying, G.G., Kookana, R.S., Kumar, A., 2008. Fate of estrogens and xenoestrogens infour sewage treatment plants with different technologies. EnvironmentalToxicology and Chemistry 27, 87e94.

Ying, G.G., Kookana, R.S., Kumar, A., Mortimer, M., 2009. Occurrence and implica-tions of estrogens and xenoestrogens in sewage effluents and receiving watersfrom South East Queensland. Science of the Total Environment 407, 5147e5155.

Ying, G.G., Williams, B., Kookana, R., 2002. Environmental fate of alkylphenols andalkylphenol ethoxylates e a review. Environment International 28, 215e226.

Zhang, Z.L., Hibberd, A., Zhou, J.L., 2006. Optimisation of derivatisation for theanalysis of estrogenic compounds in water by solid-phase extraction gaschromatography-mass spectrometry. Analytica Chimica Acta 577, 52e61.

Zhao, J.L., Ying, G.G., Chen, F., Liu, Y.S., Wang, L., Yang, B., Liu, S., Tao, R., 2011.Estrogenic activity profiles and risks in surface waters and sediments of thePearl River system in South China assessed by chemical analysis and in vitrobioassay. Journal of Environmental Monitoring 13, 813e821.

Zhao, J.L., Ying, G.G., Wang, L., Yang, J.F., Yang, X.B., Yang, L.H., Li, X., 2009. Determi-nation of phenolic endocrine disrupting chemicals and acidic pharmaceuticals insurfacewater of the Pearl Rivers in South China by gas chromatography-negativechemical ionization-mass spectrometry. Science of the Total Environment 407,962e974.