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Environmental Pollution 159 (2011) 148e156
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Environmental Pollution
journal homepage: www.elsevier .com/locate/envpol
Assessing estrogenic activity in surface water and sediment of the Liao Riversystem in northeast China using combined chemical and biological tools
Li Wang, Guang-Guo Ying*, Jian-Liang Zhao, Shan Liu, Bin Yang, Li-Jun Zhou, Ran Tao, Hao-Chang SuState Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, China
Estrogenic risks to aquatic organisms were assessed by using combine
d chemical analysis and in vitro bioassay.a r t i c l e i n f o
Article history:Received 9 December 2009Received in revised form20 June 2010Accepted 7 September 2010
Keywords:Endocrine disrupting compoundsYeast estrogen screenEstrogenicityRiverWaterSediment
* Corresponding author.E-mail address: [email protected] (G.-G.
0269-7491/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.envpol.2010.09.017
a b s t r a c t
This paper investigated some selected estrogenic compounds (4-t-octylphenol: 4-t-OP; 4-nonylphenols:4-NP; bisphenol-A: BPA; diethylstilbestrol: DES; estrone: E1; 17b-estradiol: E2; 17a-Ethinylestradiol:EE2; triclosan: TCS) and estrogenicity in the Liao River system using the combined chemical and in vitroyeast screen bioassay and assessed their ecological risks to aquatic organisms. The estrogenic compounds4-t-OP, 4-NP, BPA, E1, E2 and TCS were detected in most of the samples, with their concentrations up to52.1 2065.7, 755.6, 55.8, 7.4 and 81.3 ng/L in water, and up to 8.6, 558.4, 33.8, 7.9, <LOQ and 33.9 ng/g insediment, respectively. However, DES and EE2 were not detected in the Liao River. The estrogenequivalents (EEQ) of the water and sediment samples were also measured by the bioassay. High estro-genic risks to aquatic organisms were found in the river sections of metropolitan areas and the lowerreach of the river system.
� 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Endocrine disrupting chemicals (EDCs) as environmentalcontaminants have received considerable attention since the early90s (Jobling and Sumpter, 1993; Desbrow et al., 1998; Heberer,2002; Silva et al., 2002; Ying et al., 2002a,b, 2008). These chem-icals can interfere with the normal functioning of hormonal systemin wildlife and human beings and cause adverse effects on theorganisms (Crews et al., 2000; Damstra et al., 2002; Jobling et al.,2004). One of the widely reported effects in the aquatic environ-ment is the fish feminization in some rivers (Bortone et al., 1989;Jobling et al., 1998). Widespread sexual disruption in wild fish inthe UK has been found due to exposure to the discharges fromsewage treatment plants that contain estrogenic chemicals such asalkylphenols and hormone steroids (Jobling et al., 1998; Sumpter,1998; Tyler and Routledge, 1998). Therefore, it is important tomonitor these estrogenic compounds in riverine environments inorder to protect the ecosystem.
Various chemical and biological tools have been developed andused in the screening of estrogenic compounds in the environment,and the combinations of both tools could provide complementary
Ying).
All rights reserved.
information for contamination assessment (Desbrow et al., 1998; Fuet al., 2007; Labadie and Hill, 2007; Bicchi et al., 2009; Streck, 2009;Zhao et al., 2009). Chemical analysis using gas chromatogra-phyemass spectrometry (GCeMS) or liquid chromatogra-phyetandem mass spectrometry (LCeMS/MS) was applied todetermine target estrogenic compounds in rivers at ng/L to mg/Llevels (Kolpin et al., 2002; Kim et al., 2007; Ko et al., 2007; Labadieand Hill, 2007;Mici�c and Hofmann, 2009; Ying et al., 2009). Variousin vitro bioassays (e.g. E-SCREEN, yeast estrogen screen (YES) andestrogen responsive chemically activated luciferase (ER-CALUX))based on the interaction between chemicals and estrogenicreceptors can be used to evaluate the integrated estrogenic activityof environmental samples, without the necessity of knowing allcompounds present that contribute to the activity (Soto et al., 1995;Routledge and Sumpter, 1996; Legler et al., 2002). Combined invitro bioassay and chemical analysis could be used to better assesscausal links between effects observed in the aquatic environmentand chemical analysis results (Sumpter and Johnson, 2008; Viganòet al., 2008; Streck, 2009). However, more research is needed forthe assessment of estrogenicity in aquatic environments by usingthe combined chemical and biological tools.
The Liao River system is the largest river in northeast China,which originates from the Qilaotu Mountain and flows a distance of1345 km into the Bohai Bay. In the river basin with a population of35 million, 170 million tons of wastewaters are generated annually
L. Wang et al. / Environmental Pollution 159 (2011) 148e156 149
and only half of the wastewaters are treated (Li et al., 2000). Thesewastewaters generated in the region are mainly discharged into theriver system. Hence, water quality of the Liao River system has beenseriously deteriorated in the last 10 years. Since sewage effluentsare the major source of estrogenic compounds in the aquaticenvironment (Ying et al., 2008, 2009), it is crucial to understand thelevels and fate of these estrogenic compounds in the river systemand their potential adverse effects on aquatic organisms. To the bestof our knowledge, no published information has been available onthe estrogenic activity and the levels of estrogenic compounds inthe Liao River system.
The objective of this study is to investigate estrogeniccompounds and estrogenic activity in the Liao River system usingcombined chemical analysis and in vitro bioassay. Selected targetestrogenic compounds including 4-t-octylphenol (4-t-OP), 4-non-ylphenols (4-NP), bisphenol-A (BPA), diethylstilbestrol (DES),estrone (E1), estradiol (E2), 17a-Ethinylestradiol (EE2), and triclo-san (TCS) in the surface water and sediment were determined usingGCeMS, while estrogenic activities of surface water and sedimentsamples were measured using the YES bioassay. Potential risks toaquatic organisms were assessed based on the data from thecombined chemical analysis and YES bioassay.
2. Materials and methods
2.1. Chemicals and materials
The target compounds (4-t-OP, 4-NP, BPA, DES, E1, E2, EE2, and TCS) as well asinternal standards 4-n-nonylphenol (4-n-NP), bisphenol-A-d16 (BPA-d16), estrone-2,4,16,16-d4 (E1-d4), 13C-labelled triclosan (13C12-TCS) were purchased from Supelco(USA), Dr Ehrenstorfer GmbH (Germany), Cambridge Isotope Laboratories (USA) orRiedel-de-Haën (RDH, Germany) (Table 1).
The derivatization reagent pentafluorobenzoyl chloride (PFBOCl, purity >99%)was purchased from Aldrich. All reagents of HPLC grade used for sample processingand analysis (methanol, n-hexane, ethyl acetate, toluene, dichloromethane, trie-thylamine (TEA) and pyridine) were obtained from Merck Corporation (Shanghai,China). The cartridges used for solid phase extraction (SPE) were Oasis HLBcartridges (N-vinylpyrrolidone-m-divinylbenzene copolymer, 500 mg, 6 mL) thatwere obtained fromWaters Corporation (Milford, MA, USA). Glass fiber filters (GF/F,pore size 0.7 mm) were obtained from Whatman (Maidstone, England) and pyro-lyzed at 450 �C for 4 h prior to use. Deionized water was prepared with a Milli-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
Two sampling campaigns were carried out to collect water and sedimentsamples in the wet season (July 2008) and dry season (November 2008) along theLiao River system. During the sampling, a global position system (GPS) was used tolocate the sampling sites. Samples (water and sediment) were collected from 21 sitesin the four major watersheds of the Liao River system, including the Liaohe River,Hunhe River, Taizihe River and Daliaohe River (Fig. 1). These sites were selected
Table 1Details of the phenolic endocrine disrupting chemicals and their characteristic ionsand retention times.
Compound Supplier M.W. R.T. Ions
4-n-NP (I.S.) Dr. 220 20.78 414.2 415.24-t-OP Supelco 206 17.13 400.2 401.24-NP Dr. 220 18.15 414.2 415.2BPA-d16 (I.S.) Supelco 244 34.15 630.2 420.2BPA Supelco 228 34.34 616.1 406.1E1-d4 (I.S.) Cambridge 274 36.69 468.2 450.1DES RDH 268 35.41 656.1 446.1E1 RDH 270 36.77 464.2 418.1EE2 Dr. 296 37.38 490.2 472.1E2 Dr. 272 49.59 660.0 661.113C12-TCS (I.S.) Cambridge 301.5 25.25 494.0 496.0 299.0 301.0TCS Dr. 289.5 25.26 482.0 484.0 287.0 289.0
I.S.: internal standard; M.W.: molecular weight; R.T.: retention time.Underlined ions are ions used for quantitation.
based on suggestions from the national and local environmental protection agenciesand most of them were located at the water monitoring stations along the river.
Grab water samples were collected in 1 L clean amber glass bottles from 0.5 to1 m below the surface. Before sample collection, each bottle was pre-rinsed withriver water for three times. About 50 mLmethanol and 400 mL 4 M sulfuric acid wereadded into each bottle to preserve the samples. Then the collected water sampleswere transported in a cooler to the laboratory, and stored at 4 �C before filtration andextraction.
The sediment samples were collected with a core sampler. The sediments weresieved in the field to remove gravel and plants and stored in 1 L bottles. Sodiumazide (NaN3, 1 g) was added into each bottle to inhibit microbial growth. Afterreturning to the laboratory, the sediments were freeze-dried, homogenized ina glass mortar, passed through a 60 mesh standard sieve and stored at �18 �C untilextraction. Total organic carbon (TOC, %) of each sediment sample was measuredwith an LECO C230 carbon analyzer (USA) after removal of carbonates with HCl,while sediment particle size distributionwas analyzed by using the pipette method.
2.3. Sample extraction and derivatization
The water samples (1 L each) 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, duplicate watersamples (1 L each) were spiked with the internal standards (100 ng each of 4-n-NP,BPA-d16, and E1-d4) for chemical analysis, while another unspiked duplicate watersamples were used for yeast estrogen screen test. The SPE HLB cartridges werepreconditioned with 10 mL of methanol and 10 mL of deionized water. Watersamples were passed through the cartridges at a flow rate of 5e10 mL/min undervacuum. After dried with air for at least 1 h, the target compounds were eluted with7 mL methanol, followed by 5 mL dichloromethane. The extracts were mixed anddried under a gentle nitrogen stream, then redissolved in 1 mL of methanol. Eachfinal extract was then filtered through a 0.45 mmmembrane filter into a 2 mL amberglass vial. The vials were kept at �18 �C for later analysis.
Estrogenic compounds in the sediments were extracted by ultrasonic-assistedsolvent extraction. Similar to the spiking approach used for river water extraction,two replicate sediments were spiked with 100 ng of each internal standard forchemical analysis, and another two replicate samples were directly extracted forbioassay. Five grams of the prepared sediments were mixed with ethyl acetate(10 mL) in a 50 mL of screw-top centrifuge tube. The tube was ultrasonicated for15 min and centrifuged at 1370 g for 10 min, and the supernatant was collected inanother tube. The sediments were extracted for two additional times with 10 mLand 5 mL of solvent ethyl acetate, respectively. The supernatants were combined andwere concentrated to about 1e2 mL on a rotary evaporator. The extract was furtherpurified with a glass column (6 mm i.d.) loaded with 1 g of silica gel. The elutionwascarried out using 6 mL ethyl acetate. The eluate was concentrated to nearly drynessunder a gentle nitrogen stream and redissolved in 1 mL methanol for furthertreatment and later analysis. The purified water and sediment extracts werederivatized with PFBOCl (Zhao et al., 2009) prior to the analysis by GCeMS withselected ion monitoring mode (SIM).
2.4. Instrumental analysis
The target compounds were analyzed using an Agilent 6890N gas chromato-graph (Agilent, USA) equipped with an Agilent 5975B MSD mass spectrometer witha chemical ionization (CI) source. An HP-5MS GC capillary column (30 m, 0.25 mmi.d., 0.25 mm film thickness) was used for separation of all target compounds. Heliumwas used as the carrier gas and maintained at a constant flow rate of 1.0 mL/min forthe analysis of the target compounds. A sample volume of 2 mL was injected in thesplitless mode at an inlet temperature of 300 �C. The GC oven temperature was keptat 80 �C for 1 min, followed by the first ramp at 10 �C/min to 220 �C, second ramp at4 �C/min to 260 �C, third ramp at 5 �C/min to 300 �C, then to 310 �C at 20 �C/min andheld at 310 �C for 15 min. The MS interface temperature was maintained at 310 �C.The analytical method for the target compounds has been described in details byZhao et al. (2009). Ions selected for GCeMS analysis are given in Table 1. Positiveidentification of each target compound was based on acceptance criteria of itsretention time and confirmation ions (difference within 20%).
2.5. Yeast estrogen screen bioassay
The recombinant yeast for bioassay was kindly provided by J.P. Sumpter (BrunelUniversity, Uxbridge, UK). The yeast estrogen screen test was carried out asdescribed by Routledge and Sumpter (1996) with a few modifications. Briefly, eachextracted samplewas 2-fold diluted in 12 series on a row of a 96-well microplate (BDFalcon�, USA) with the dilution factor of 2 using methanol, then 10 mL of eachconcentration was transferred to corresponding well on another 96-well test plate.The methanol in each well was allowed to dry absolutely in a laminar flow cabinet.The preparations for growth media and assay media were the same as reported byRoutledge and Sumpter (1996). 200 mL of seeded assay media contained recombi-nant yeast solution and chlorophenol red-b-D-galactopyranoside (CPRG) was addedto eachwell on test plate. The test platewas sealed and packedwith foil, then shaken
Fig. 1. Location map for the Liao River system in northeast China.
L. Wang et al. / Environmental Pollution 159 (2011) 148e156150
vigorously for 5 min on a microplate shaker and incubated statically at 32 �C indarkness. After 24 h the test plate was shaken for 10 min at 500 rpm. After 72 hincubation, the plate was again shaken for 10 min at 500 rpm, and left for 1 h toallow the yeast cells to settle down, and the absorbance at 620 nm and 540 nmwasmeasured on a BMG microplate reader (BMG Lab technologies, Offenburg,Germany). E2 was used as the positive control with a concentration froma maximum 2.72 ng L�1 (1�10�8 M) to a minimum 1.33 ng L�1 (5�10�12 M) inseries diluted wells, and 2 rows of 10 mL methanol only in each well were used asnegative control (also dried in laminar flow cabinet after added). The absorbancemeasured for serially diluted standard E2 and sample extracts was fitted to fourparameter logistic curves. The total estrogenic activity in the environmental sampleswas measured by comparing to the activity of the natural estrogen, E2, andexpressed as estradiol equivalent (EEQ). The reporting limit for the YES bioassay was0.2 ng/L in water and 0.04 ng/g in sediment based on spiking experiments.
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 (Thorpe et al., 2006; Bicchi et al.,2009). The calculated EEQ was expressed as the sum of all estrogenic contribu-tions of seven compounds by multiplying their corresponding relative potency (RP)(Table 2) and chemical concentration.
Table 2Relative potencies (RPi) of the seven target compounds.
Compound Abbreviation RPa
4-tert-octylphenol 4-t-OP 0.000934-nonylphenol 4-NP 0.00063bisphenol-A BPA 0.00011estrone E1 0.3diethylstilbestrol DES 0.83estradiol E2 1.017a-ethinylestradiol EE2 2.2
a RP: relative potency compared with E2.
2.6. 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. In the samples analyzed, recoveries ofthe surrogate standards were mostly more than 70%. Relative recoveries of the sixestrogenic compounds using the internal standards 4-n-NP, BPA-d16, E1-d4and 13C12-TCS ranged from 75 to 145% for the water samples at the spikedconcentration of 100 ng/L, and from 75 to 106% for the sediment samples at thespiked concentration of 100 ng/g (Zhao et al., 2009). Only trace amount of 4-NP wasfound in procedural blanks; hence the background values were not subtracted from
the sample measurements. The limit of detection (LOD) and limit of quantitation(LOQ) of the target compounds were calculated based on the standard derivations(SD) of seven replicates of spiked reservoir water and sediment at the concentrationof 5 ng/L and 10 ng/g. LOD is defined as three times of SD, and LOQ is 10 times of SD.The limits of quantification (LOQ) for 4-t-OP, 4-NP, BPA, DES, E1, E2, EE2, and TCS insurface water were 1, 7, 2, 0.5, 0.5, 1, 0.7, and 0.5 ng/L, respectively, while those insediment were 0.9, 4.9, 2.6, 2.3, 1.1, 3.5, 2.5, 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
Six target estrogenic compounds (4-NP, 4-t-OP, BPA, E1, E2 andTCS) were detected in surface water from the Liao River system in
L. Wang et al. / Environmental Pollution 159 (2011) 148e156 151
both wet (July) and dry (November) seasons (Fig. 2), but DES andEE2 were found to have concentrations below the LOQs. Theconcentrations for 4-NP, BPA and TCS in the surface water sampleswere in general higher than those for 4-t-OP, E1 and E2. The levelsof 4-NP were the highest at all sites, while those for E2 were thelowest. The measured concentrations for the target compounds inthe dry season were always higher than in the wet season. Forexample, the concentrations of 4-NP in surface water in July rangedfrom 126.3 to 900.7 ng/L with a median of 178.7 ng/L, while thosein November ranged from 417.8 to 2065.7 ng/L with a median of599.3 ng/L. The concentrations of E1 in the water from Julysampling ranged from<LOQ to 15.2 ng/L with a median of 1.5 ng/L,while those from November sampling ranged from 1.4 to 55.8 ng/Lwith a median of 4.9 ng/L.
These target compounds varied widely spatially, with highconcentrations being found more frequently at sites 6, 10, 11, 12, 19,20 and 21 (Table S3 and Table S4, Supplementary). From the data forthe November sampling, the highest concentrations for all sixcompounds in surface water were found in site 19. These sites aremainly located in metropolitan areas and lower reaches of the LiaoRivers (Fig. 1).
The six estrogenic compounds (4-NP, 4-t-OP, BPA, E1, E2 andTCS) were detected in the sediments of the Liao River system; butE2 was only detected in some sites and below its LOQ. DES andEE2 were not detected in the river sediments, thus in the followingonly six detected compounds were discussed. E1 was determined
Compounds
4-t-OP 4-NP BPA E1 E2 TCS
)L/
gn
(n
oi
ta
rt
ne
cn
oC 0
5
10
15
400
800July 2008
100% 100% 100% 95% 29% 100%
Compounds
4-t-OP 4-NP BPA E1 E2 TCS
)L/
gn
(n
oi
ta
rt
ne
cn
oC
0
25
50
75
500
1000
1500
2000
November 2008
100% 100% 100% 100% 43% 100%
Fig. 2. Box plots for the estrogenic compounds (ng/L) in surface water of the Liao Riversystem. The estrogenic compounds detected in the water were: 4-t-octylphenol (4-t-OP), 4-nonylphenols (4-NP), bisphenol-A (BPA), estrone (E1), estradiol (E2) and tri-closan (TCS). The horizontal lines represent 5th, 50th, median and 95th percentiles,and the boxes represent 25th and 75th percentiles. Median and mean concentrationsare displayed as solid and dashed horizontal lines, respectively. Outliers are shown asindividual points. The percentage value below each box is the detection of thecompound.
at concentrations ranging between <LOQ and 7.9 ng/g witha median of <LOQ in July and between <LOQ and 3.7 ng/g witha median of <LOQ in November (Fig. 3; Table S7, Supplementary).Highest concentrations were detected for 4-NP, ranging between10.0 and 375.4 ng/g with a median of 22.3 ng/g in July andbetween 17.8 and 558.4 ng/g with a median of 49.1 ng/g inNovember. Similar patterns for the six estrogenic compounds werefound in the two sampling events with the median concentrationsin the decreasing order: 4-NP> BPA> TCS> E1>4-t-OP> E2.Spatially, higher concentrations for the estrogenic compoundswere often observed in sites 8e12 and 19e21 (Table S5 and TableS6, Supplementary).
The (pseudo-) partitioning coefficients (Kd) for the estrogeniccompounds were calculated as the ratio of concentrations in sedi-ment to water phase. The Kd values for 4-t-OP, 4-NP, BPA, E1 andTCS correlated positively and significantly with the TOC (%) insediment (p< 0.05) (Fig. 4). From the R2 values in the graphs, it canbe seen that these correlations between the Kd and TOC are weakfor 4-NP, BPA, E1 and TCS, but medium for 4-t-OP. It is also foundthat there are significant but weak correlations between the Kd andclay content (%) in sediment for the three estrogenic compounds E1,BPA and TCS (p< 0.05), but no significant correlations for 4-t-OPand 4-NP (Fig. S1, Supplementary).
Compounds
4-t-OP
)g
/g
n(
no
it
ar
tn
ec
no
C
0
10
20
150
300
450
July 2008
5% 100% 74% 37% 58%
Compounds
4-t-OP
)g/
gn
(n
oi
ta
rt
ne
cn
oC
0
5
10
15
20
200
400
600
November 2008
24% 100% 86% 24% 62%
TCSE1BPA4-NP
TCSE1BPA4-NP
Fig. 3. Box plots for the estrogenic compounds (ng/g dw) in sediment of theLiao River system. The estrogenic compounds detected in the sediment were: 4-t-octylphenol (4-t-OP), 4-nonylphenols (4-NP), bisphenol-A (BPA), estrone (E1) andtriclosan (TCS). The horizontal lines represent 5th, 50th, median and 95th percen-tiles, and the boxes represent 25th and 75th percentiles. Median and meanconcentrations are displayed as solid and dashed horizontal lines, respectively.Outliers are shown as individual points. The percentage value below each box is thedetection of the compound.
TOC (%)
0 2 4 6 8 10 12
)g
K/
L(
dK
0
100
200
300
400
500
600
4-t-OP y=34.1x-11.5
R2
=0.4093
F=28 p<0.0001
TOC (%)
0 2 4 6 8 10 12
)g
K/
L(
dK
0
100
200
300
400
500
600
700
4-NP y=32.2x+99.1
R2
=0.1936
F=10 p=0.0035
TOC (%)
0 2 4 6 8 10 12
)g
K/
L(
dK
0
50
100
150
200
250
300
350
BPA
y=14.9x+51.2
R2
=0.3425
F=5 p=0.0264
TOC (%)
0 2 4 6 8 10 12
dK
(L
K/
g)
0
2000
4000
6000
8000
10000
E1
y=232.0x+143.6
R2
=0.1101
F=5 p=0.0318
TOC (%)
0 2 4 6 8 10 12
)g
K/
L(
dK
0
1000
2000
3000
4000
5000
TCS
y=183.0x+58.6
R2=0.2763
F=15 p=0.0004
Fig. 4. Plots of the distribution coefficients (Kd, L/kg) of the estrogenic compounds versus the total organic carbon content (TOC, %) of the sediments. The estrogenic compoundsdetected in the sediment were: 4-t-octylphenol (4-t-OP), 4-nonylphenols (4-NP), bisphenol-A (BPA), estrone (E1) and triclosan (TCS).
L. Wang et al. / Environmental Pollution 159 (2011) 148e156152
3.2. Estrogenicity in surface water and sediment
The estrogenicity in surface water measured by the YES assay inNovember (dry season) was mostly higher than that measured inJuly (wet season) (Table 3). Only nine sites (S6e8, S11e12, andS18e21) out of 21 sites were found in the wet season to have theEEQ above the reporting limit (0.2 ng/L), with the highest concen-tration up to 17.52 ng/L at site 11. In the dry season, only three sites(S13e15) were found to have the EEQ values below the limit ofdetection. Higher EEQ values (>3 ng/L) were determined at sitesS8e9, S11e12 and S18e20, with the highest value (78.83 ng/L) atsite 19.
Estrogenic activities in the sediments from the Liao River systemwere observed in some sites in both seasons (Table 3). The EEQvalues in the wet season ranged from <LOQ to 4.76 ng/g, whilethose in the dry season ranged from <LOQ to 6.04 ng/g. The EEQvalues in the sediments were highly variable among the sites(Fig. 5). Higher EEQ values were found at sites 6, 9, 10, 15 and 20with the highest concentration of 6.04 ng/g at site 9. The EEQ valuesin the sediments from the Liao River system correlated positivelyand significantly with the TOC (%) in the sediments (p< 0.01), but
there were no significant correlation between the EEQ values andclay content (%) in the sediments (p¼ 0.2475) (Fig. 6).
4. Discussion
4.1. Distribution of estrogenic compounds
The concentration ranges for the six estrogenic compounds insurface water mostly fall within those reported in the literature(Table 4). The alkylphenol concentrations in the Pearl Rivers werefound up to 2470 ng/L for 4-t-OP and 8890 ng/L for 4-NP (Zhaoet al., 2009), which are much higher than those in the Liao Rivers.The concentration ranges for the estrogenic compounds in sedi-ment for the Liao Rivers are comparable to those previouslyreported in the literature (Table 4). Estrone (E1) and xenoestrogens(4-NP and 4-t-OP) were also detected in composite sedimentsamples from the middle River Po, but E2 and BPA were not foundin the sediments (Viganò et al., 2006).
Seasonal and spatial variations in concentrations of the sixestrogenic compounds were observed in surface water of the LiaoRiver system. The concentrations of 4-t-OP, 4-NP, BPA and E1 in the
Table 3Estrogen equivalent values (EEQ) for the water and sediment samples of the Liao River system in July and November 2008.
Sites Water in July Water in November Sediment in July Sediment in November
Predicted EEQ(ng/L)
Determined EEQ(ng/L)
Predicted EEQ(ng/L)
Determined EEQ(ng/L)
Predicted EEQ(ng/g)
Determined EEQ(ng/g)
Predicted EEQ(ng/g)
Determined EEQ(ng/g)
S1 0.52 NDa 2.22 1.79 0.0014 <LOQb 0.0044 <LOQS2 0.37 ND 2.02 1.06 0.0011 <LOQ 0.0037 0.12S3 0.57 ND 2.21 1.17 0.0011 ND 0.0020 <LOQS4 0.36 ND 1.01 0.58 NAc NA 0.0038 <LOQS5 0.32 ND 4.61 1.43 0.0023 <LOQ 0.0033 0.05S6 2.13 0.36 0.88 0.55 0.0029 <LOQ 0.0043 1.38S7 0.82 0.58 0.91 0.26 0.55 0.14 1.15 0.41S8 1.01 0.82 7.22 6.13 3.96 4.76 1.87 0.33S9 0.31 <LOQ 11.26 6.25 2.21 1.68 0.016 6.04S10 0.74 ND 1.47 1.04 0.81 0.28 1.62 1.50S11 13.64 17.52 10.71 5.18 0.0013 ND 0.0022 0.11S12 6.73 4.68 10.17 6.19 0.0056 ND 0.76 0.12S13 0.02 ND 0.77 ND 0.95 0.17 0.0018 <LOQS14 0.42 ND 1.11 ND 0.55 0.27 0.0054 0.07S15 0.26 ND 1.01 ND NA NA 0.011 5.85S16 0.77 ND 4.40 2.47 0.0012 ND 0.016 <LOQS17 0.88 <LOQ 5.69 2.26 0.0025 <LOQ 0.0070 0.05S18 1.77 1.72 15.74 18.31 0.0024 ND 0.0037 0.05S19 6.99 9.44 35.54 78.83 0.0024 <LOQ 0.014 0.26S20 4.08 2.23 12.24 11.09 1.44 0.25 0.76 1.00S21 3.89 2.80 2.58 0.67 0.017 0.72 0.0088 0.08
a ND: not detected.b <LOQ: below the reporting limit.c Not available.
L. Wang et al. / Environmental Pollution 159 (2011) 148e156 153
dry season (November, winter) were generally higher than those inthe wet season (July, summer). Ko et al. (2007) also measuredhigher concentrations in treated sewage effluents and river watersof Korea in winter than in summer. This is mainly due to the effects
July 2008 November 2008
)L/
gn
(Q
EE
de
ni
mr
et
eD
0
5
10
15
50
100
43% 86%
July 2008 November 2008
)g/
gn
(Q
EE
de
ni
mr
et
eD
0
1
2
4
6
8
42% 76%
Fig. 5. Estrogenicity (EEQ) of the water and sediment samples measured by the yeastestrogen screen bioassay (YES). The horizontal lines represent 5th, 50th, median and95th percentiles, and the boxes represent 25th and 75th percentiles. Median and meanconcentrations are displayed as solid and dashed horizontal lines, respectively. Outliersare shown as individual points. The percentage value below each box is the detectionof the compound.
caused by dilution and temperature change. In the Liao River basin,rainfall (75% of annual total rainfall) is concentrated in June toSeptember every year. In November, the temperature in the regiondrops to minus degrees, which can reduce the dissipation of
TOC (%)
0 2 4 6 8 10 12
)g/
gn
(Q
EE
de
ni
mr
et
eD
0
1
2
3
4
5
6
7
y=0.6x-0.2
R2
=0.6771
F=84 p<0.0001
Clay (%)
0 5 10 15 20 25
)g/
gn
(Q
EE
de
ni
mr
et
eD 0
1
2
3
4
5
6
7
y=0.1x+0.4
R2
=0.0333
F=1 p=0.2475
Fig. 6. Plots of the measured EEQ values (ng/g) versus the total organic carbon content(TOC, %) and clay content (%) in the sediments.
Table 4Comparison of the estrogenic compounds in water and sediment.
Sample Location Concentration (ng/L) Referencec
4-t-OP 4-NP BPA E1 E2 TCS
Water China 34.2e599 122.4e66.2 (34.7)a 1.4e33.9 (14.4) 2
1.0e2470 28.1e8890 2.2e1030 ND-75 ND-7.5 0.6-347 3Korea NDb ND-244.8 ND-39.4 4
1.7e5.0 (3.6) ND ND 5Japan ND-81.9 ND-147.0 ND-76.3 ND-85.6 ND-12.3 6Austria ND-41 ND-890 (31) ND-600 ND-4.6 (0.58) ND-1.2 (0.19) 7
13e94 (29) 367e1053 (676) 4e59 (14) 1.1e20.9 (5.08) 0.54e3.77 (1.54) 8USA 0e147.2 0-4.7 0-4.5 8.8e34.9 9
ND-40000 Nd-12000 Nd-112 ND-200 ND-2300 10Italy <0.5-211 <1.0e145 <1.2e10 <1.0e36 11Spain 500e15,000 (8) ND 12Liao River system 2.3e52.1 (6.6) 122.4e2065.7 (536.9) 12.3e755.6 (149.8) ND-55.8 (6.5) ND-7.4 (1.0) 6.5e81.3 (28.4) This study
Sample Location Concentration (ng/g)
Sediment Italy 47e192 <2.0e118 12UK 0.40e3.30 <0.03e1.20 13Liao River system <LOQ-8.6 10.0e558.4 (74.5) <LOQ-33.8 (5.3) <LOQ-7.9 <LOQ <LOQ-33.9 (5.1) This study
a Concentration range: minimum to maximum (mean).b ND: not detected.c 1. Xu et al., 2006; 2. Chen et al., 2007; 3. Zhao et al., 2009; 4. Ko et al., 2007; 5. Kim et al., 2007; 6. Furuichi et al., 2004; 7. Hohenblun et al., 2004; 8. Ying et al., 2009; 9. Zhang
et al., 2007; 10. Kolpin et al., 2002; 11. Pojana et al., 2007; 12. Petrovic et al., 2002; 13. Labadie and Hill, 2007.
Predicted EEQ from chemical analysis (ng/L)
0 10 20 30 40
)
L
/
g
n
(
Q
E
E
d
e
n
i
m
r
e
t
e
D
-20
0
20
40
60
80
100
y=1.0x-0.8
R 2
=0.8515
F=223.6 p< 0.0001
water
EEQ calculated from chemical analysis (ng/g)
0 1 2 3 4 5
)
g
/
g
n
(
Q
E
E
d
e
n
i
m
r
e
t
e
D
0
1
2
3
4
5
6
7
y=0.8x
R 2
=0.6868
F=83 p <0.0001
Sediment
Fig. 7. Plots of the EEQ values measured by YES versus the EEQ values calculated fromchemical analysis of the water and sediment samples.
L. Wang et al. / Environmental Pollution 159 (2011) 148e156154
chemicals in water. Spatial variations were also found for the sixestrogenic compounds based on the chemical analysis and YESbioassay. Higher concentrations weremostly detected in sites 8e12and 18e21, which are associated with large metropolitan areassuch as Fushun, Shenyang and Anshan, and lower reach of theDaliaohe River.
Once a chemical enter an aquatic environment, partitioning ofthe chemical between water and sediment phases becomes animportant process. Due to their moderate hydrophobic nature,partitioning of the estrogenic compounds investigated in thepresent study occurred in the rivers. Significant correlationsbetween the Kd values and TOC (%) have been observed for the sixestrogenic compounds except for E2, which was below the quan-tification limit. This is probably due to rapid degradation or trans-formation of E2 in the aquatic environment (Ying et al., 2002b,2008). Significant and positive correlation was also foundbetween the EEQ and TOC, suggesting that estrogenic compoundshave a tendency to adsorb onto sediment. Adsorption of a hydro-phobic compound onto sediment is mainly related to sedimentproperties such as TOC and clay contents in addition to its ownphysicochemical properties (Baker et al., 1991; Zhu et al., 2008).Hence sediments with higher TOC in the Liao Rivers are more likelyto have higher accumulation of the target estrogenic compounds,such as sites 7e10 with their TOC ranging from 1.8 to 5.7% (Table S1,Supplementary).
In addition to the direct partitioning of the dissolved phasefraction of the target compounds from the bulk river water to theriver bed sediment, these compounds also have a propensity topartition to particulate organic matter in the river water. Therefore,subsequent settling and accumulation of this particulate fraction toriver sediments will largely control the mass flux of thesecompounds within the sediments.
4.2. Comparison of estrogenicity by YES bioassay and chemicalanalysis
The theoretical estrogenicity was calculated as the summationof individual estrogenicity estimated from each chemical concen-tration monitored by GCeMS (Bicchi et al., 2009). The theoreticalestrogenicity calculated based on chemical analysis (Calculated
EEQ) had significant correlation with the total estrogenicitymeasured by the YES bioassay (Determined EEQ) for surface waterand sediment in the Liao River system (Fig. 7). One data point forthe water samples and two data points were removed as theoutliers in the correlation analysis. For most water samples, the EEQvalues determined by the YES assay were lower than those calcu-lated from chemical analysis (Table 3). This can be explained by thepresence of unknown antagonists in the water samples (Tanakaet al., 2001; Witters et al., 2001). Some water extracts exerted
L. Wang et al. / Environmental Pollution 159 (2011) 148e156 155
toxicity to the yeast cells during the bioassay, which affecteddevelopment of color and further measurement of EEQ. Similartoxic effects on yeast cells have been reported previously inwastewater (Tanaka et al., 2001) and surface water (Witters et al.,2001).
For sediment samples, the EEQ values determined by the YESbioassay were different from those calculated from chemicalanalysis (Fig. 7). Hence the selected estrogenic compoundsaccount for majority of the estrogenicity in the Liao River system.But the measured EEQ values for the water samples from site 11and site 19 and for the sediment samples from site 9 and site 15were higher than those calculated values (Table 3). This suggeststhat some unknown chemicals might contribute to the estro-genicity of the samples (Soto et al., 2004; Sarmah et al., 2006;Viganò et al., 2008). The present study demonstrates that estro-genic activity in a sample may not be fully explained by chemicalanalysis of the selected target compounds, and an estrogenicscreening bioassay such as the YES assay could provide the overallestrogenicity of a sample. Therefore, the EEQ measured by the YESin the present study could be used in the further risk assessmentfor the Liao River system.
4.3. Risk assessment
The presence of estrogenic compounds and their estrogenicactivities might affect aquatic organisms such as fish in the rivers bydisrupting the normal hormonal functions of these organisms(Jobling et al., 1998; Sumpter, 1998; Tyler and Routledge, 1998). Thereported lowest observed effect concentration (LOEC) values forestrogenic responses in medaka, trout and roach are 10 ng/L for E1and E2 (Metcalfe et al., 2001; Routledge et al., 1998). Young et al.(2002) proposed a tentative long term predicted no-effect-concentration (PNEC) for freshwater life of 1 ng/L for E2 based ona thorough literature review. This means that EEQ values> 1 ng/Lin the aquatic environment might cause reproductive problems insome fish. According to this PNEC (1 ng/L), 6 sites in the wet seasonand 14 sites in the dry season have the potential to cause estrogeniceffects on some aquatic organisms (Table 3). High risks have beenfound in sites 18, 19 and 20 with the EEQ values more than 10 ng/L.Moreover, estrogenic activities in these sites were mainly attrib-uted to the presence of the selected estrogenic compounds (alkyl-phenols and estrogens) as shown in chemical analysis. However,a further biological exposure study is needed to establish the realconsequences of estrogenic activity in the rivers.
5. Conclusions
This study clearly demonstrated the presence of estrogeniccompounds and estrogenicity in the Liao River system using thecombined chemical and biological analyses. Based on the analyticaldata for surface water and sediment, it was found that theseestrogenic compounds have tendency to adsorb onto sediment dueto their moderate hydrophobic nature. The EEQ values in sedimentcorrelated significantly with the TOC. The selected estrogeniccompounds account for the majority of the estrogenic activitiesdetermined in the surface water and sediment samples from theriver system according to the comparative analysis of the measuredand calculated EEQ values. Estrogenic risks are expected for somesites based on the measured EEQ values in the surface water of theLiao River system. High risks are associated with those sites locatedin the metropolitan areas and lower reach of the river system.Proper measures need to be taken to reduce the discharge ofeffluents containing estrogenic compounds into the river system inorder to protect aquatic organisms in the rivers.
Acknowledgements
The authors would like to acknowledge the financial supportfrom the National Natural Science Foundation of China (NSFC40688001, 40771180 and 40821003) and Guangdong ProvincialNatural Science Foundation (8251064004000001). We also wouldlike to thank MEWang and QX Zhou for their assistance in the fieldsampling. This is a contribution no. 1246 from GIG CAS.
Appendix. Supplementary information
Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.envpol.2010.09.017.
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