Environmental Pollution 244 (2019) 462e468

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

Endocrine disrupting chemicals in wild freshwater fishes: Species,

tissues, sizes and human health risks*
Xinyi Zhou a, Zhaoguang Yang a, b, Zhoufei Luo a, Haipu Li a, b, *, Guoyao Chen a
a
Center for Environment and Water Resources, College of Chemistry and Chemical Engineering, Central South University, Changsha, 410083, PR China
b
Key Laboratory of Hunan Province for Water Environment and Agriculture Product Safety, Changsha, 410083, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Increasing attention has been devoted to the adverse effects of endocrine disrupting chemicals (EDCs) on
Received 12 June 2018 aquatic environments, such as water, sediment and sludge. To date, few studies have investigated the
Received in revised form bio-accumulative characteristics of EDCs in different tissues of diverse wild freshwater fish species and
19 September 2018
their combined impacts on human health. Five EDCs were investigated in the muscle, liver, gill and,
Accepted 4 October 2018
Available online 8 October 2018
especially, gonad of three fish species collected from the Xiangjiang River, southern China. Carnivorous
Siniperca Chuatsi or omnivorous Cyprinus Carpio accumulated higher contents of bisphenol A (BPA) and
estrone than herbivorous Parabramis Pekinensis in muscle. Furthermore, 4-n-nonylphenol and estrone
Keywords:
Endocrine disrupting compounds
were found at higher levels and more frequently in the liver, implying that the liver played an important
Aquatic organism role in basic metabolism for accumulation, biotransformation and excretion of EDCs. Highest concen-
Tissues trations of BPA found in the gonad revealed that the BPA may pose a serious threat to the reproductive
Health risk assessment system of aquatic organisms. The mean liver/muscle concentration ratios of 4-n-nonylphenol, BPA,
Correlation analysis estrone and 17a-ethynyl estradiol confirmed the prolonged exposure of the fish to these EDCs. In
addition, the relationships between the fish sizes and the EDC concentrations analyzed by Pearson
correlation analysis implied that the bioaccumulation of diethylstilbestrol and BPA increased with the
growth of Parabramis Pekinensis, and there was a balance between the uptake rate and elimination rate of
EDCs in Siniperca Chuatsi and Cyprinus Carpio. Most importantly, the cumulative impacts of combined
EDCs on human health by fish consumption were evaluated. The total estradiol equivalent quantity of
estrogens was higher than that of phenols. Also, based on the results of the Monte-Carlo simulation, the
95th percentile values of the total estimated daily intakes from consuming the three freshwater fish
species from the Xiangjiang River were higher than the acceptable daily intake.
© 2018 Elsevier Ltd. All rights reserved.

1. Introduction 2016), such as vitellogenesis, hermaphrodism and feminization in

Endocrine disrupting chemicals (EDCs) are defined as exoge-
nous agents that interfere with the normal functioning of the
endocrine system of animals and human beings (Scholz and Klüver,
2009). The EDCs in the aquatic environment have been widely
studied around the world in recent decades because of their po-
tential health risks to aquatic organisms and humans (Gong et al.,
* commonly found in food containers, certain dental fillings and
This paper has been recommended for acceptance by Maria Cristina Fossi.
* Corresponding author. Center for Environment and Water Resources, College of
Chemistry and Chemical Engineering, Central South University, Changsha, 410083,
PR China.
E-mail addresses: zhouxinyi@csu.edu.cn (X. Zhou), zgyang@csu.edu.cn (Z. Yang),
zhoufei.luo@csu.edu.cn (Z. Luo), lihaipu@csu.edu.cn (H. Li), lisboacgypt@csu.edu.cn
(G. Chen).
fish and prostate, breast and testicular cancer in humans (Liu et al.,
2011). Among these EDCs, phenols and estrogens with estrogen
properties have attracted over-whelming attention. 4-n-non-
ylphenol (4-n-NP) is the main degradation product of alkylphenol
polyethoxylates, which have been used extensively in industry,
agriculture and daily life (Wang et al., 2012). Remarkably, 4-n-NP is
more estrogenic, toxic, lipophilic and persistent than its parent
substances (Wang et al., 2016). Bisphenol A (BPA), which is mainly
used to produce polycarbonate plastic and epoxy resins, is
other materials (Gyllenhammar et al., 2012; Natalie et al., 2010). In
addition, estrogen estrone (E1) is naturally derived from the excreta
of livestock and humans (Parkkonen et al., 2000). The most potent
man-made EDCs are diethylstilbestrol (DES) and 17a-ethynyl
estradiol (EE2) (Dan et al., 2017). DES is a drug used to prevent

https://doi.org/10.1016/j.envpol.2018.10.026
0269-7491/© 2018 Elsevier Ltd. All rights reserved.
 X. Zhou et al. / Environmental Pollution 244 (2019) 462e468
miscarriages and for animal fattening in the animal breeding in-
dustry (Qiao et al., 2016). EE2 is known as the main component of
contraceptive drugs and is one of common hormone medications
for livestock and aquaculture activity (Dan et al., 2017; Ye et al.,
2013). Though phenols and estrogens have been detected in wa-
ter and sediment around the world (Gong et al., 2016), their
occurrence in fish has been reported in only a small number of
studies, and their bioaccumulation characteristics have not been
thoroughly elucidated to date.
Fish species are considered bioindicators of aquatic pollution
because of their relatively large sizes, long lives and their various
trophic levels (Ahmed et al., 2015). At the top of the food chain in
the aquatic ecosystem, fish can accumulate a considerable amount
of EDCs through gill, oral intake and surface skin (Jia et al., 2017).
Fish are widely consumed by human beings due to their high
nutritional value (Jia et al., 2017). Consumption of fish contami-
nated by EDCs might increase health risks for human beings (Jia
et al., 2016). Therefore, it was extremely important for human
health to determinate and investigate the EDC contents in fish
species.
At present, the muscle has been used as the main research ob-
ject to study EDC contamination in fish because it is the principal
part of human consumption (Dan et al., 2017; Diao et al., 2017).
Actually, it is equally important to examine other tissues such as the
liver, gill and gonad. A fish's gill was one of the major routes for the
uptake of contaminants due to their wide surface and direct and
continuous contact with the external medium (Guilherme et al.,
2008; Mieiro et al., 2009). The liver playes a central role in basic
metabolism and is the main site for accumulation, biotransforma-
tion and excretion of contaminants (Huang et al., 2013). The gonad
is a mixed gland that produces the sex cells and sex hormones. The
concentrations of EDCs in the gonad can directly reflect the
contamination of the reproductive system. In addition, the liver and
gill would be helpful to identify the exposure mode of the con-
taminants to fish, such as the dietary and/or water exposure (Jia
et al., 2016). Therefore, it is more comprehensive to determine
EDC contaminations in multiple tissues instead of only muscle.
Moreover, to the best of our knowledge, few studies have reported
the comparison of the bioaccumulations of phenols and estrogens
in different fish species with different sizes and different feeding
behaviors.
Xiangjiang River, the longest river in Hunan province, southern
China, flows through numerous villages, several industrial areas
and certain cities, such as Changsha, Xiangtan and Zhuzhou, and
received large amounts of industrial wastewater and living sewage
water. This river was polluted by industrial additive phenols (4-n-
NP and BPA) and estrogens (DES, E1 and EE2) from agriculture and
living wastewater, and the fish were exposed to EDCs.
Thus, three wild freshwater fish species were collected from the
Xiangjiang River near Changsha city and used to evaluate the dis-
tribution characteristics of the five EDCs in their muscle, liver, gill
and gonad tissues. To the best of our knowledge, the EDC concen-
trations in gonads have not been reported to date, although EDCs
were widely considered to interfere with the reproductive systems
of aquatic organisms. Furthermore, the relationships between the
analyzed EDCs and fish sizes among different fish species were
investigated. Finally, the combined impacts of targeted EDCs on
human health by fish consumption in this area were estimated in
order to control and minimize the health risks.
2. Materials and methods
2.1. Sampling
A total of 47 fish samples were purchased from professional
463
fishermen in the Xiangjiang River near Changsha city between May
and June in 2017. The fishes included 21 Parabramis Pekinensis
(P. Pekinensis, body length 27.0 ± 3.2 cm and body weight
0.27 ± 0.09 kg), 13 Cyprinus Carpio (C. Carpio, body length
36.0 ± 7.0 cm and body weight 0.92 ± 0.51 kg) and 13 Siniperca
Chuatsi (S. Chuatsi, body length 25.3 ± 2.9 cm and body weight
0.27 ± 0.10 kg). These three species are the most common edible
species in the river and belong to categories of herbivorous,
omnivorous and carnivorous fish, respectively. The fish samples
were immediately dissected after recording live weights and
lengths. The muscles, livers, gills and gonads were collected,
washed with ultra-pure water, dried with filter paper, homoge-
nized and then stored at
20  C for further extraction.
2.2. Chemicals and sample preparation
In total, five EDCs, including two phenols (4-n-NP and BPA) and
three estrogens (E1, EE2 and DES) were selected for this study and
determinated in a simultaneous method with two internal stan-
dards (bisphenol A-d16 and 17b-Estradiol-2,4-d2). The details
regarding chemicals and materials and samples preparation were
described in the Text 1 and Text 2 of Supplementary materials.
Briefly, homogenized tissue was extracted with acetonitrile by
ultrasonic extraction for 30 min. After that step, 2.0 g anhydrous
MgSO4 and 0.5 g NaCl were added, and the tube was centrifuged.
The supernatant was transferred to a polypropylene tube contain-
ing 200 mg PSA, 200 mg C18 and 600 mg MgSO4, and was centri-
fuged. Next, the supernatant was transferred into a glass centrifuge
tube and mixed with internal standards. The mixture was evapo-
rated to dryness by N2 gas. After derivatization, the extracts were
dried under N2 gas and redissolved for analysis.
2.3. GC-MS analysis and QA/QC
The targeted analytes were analyzed by Agilent 7890A-5975C
Gas Chromatography-Mass Spectrometer (GC-MS, Agilent Tech-
nologies, USA) in the selected ion monitoring (SIM) mode. More
details regarding instrumental analysis can be found in the Text 3 of
Supplementary materials.
Quality assurance and quality control (QA/QC) were performed
to ensure efficiency and reproducibility in the analytical procedure.
The method detection limits (MDLs) of the analytes in muscle, liver,
gill and gonad ranged from 0.09 to 0.50 ng/g ww, 0.03 to 1.88 ng/g
ww, 0.23 to 1.30 ng/g ww and 0.12 to 1.95 ng/g ww, respectively.
The method quantitation limits (MQLs) of the analytes in muscle,
liver, gill and gonad ranged from 0.31 to 1.67 ng/g ww, 0.11
to 6.25 ng/g ww, 0.76 to 4.32 ng/g ww and 0.40 to 7.72 ng/g ww,
respectively. No targeted EDC was detected in the procedural
blanks. The recoveries in muscle, liver, gill and gonad ranged from
66.6% to 117%, 64.6% to 122%, 47.0% to 128% and 53.2% to 103%,
respectively. The relative standard deviations (RSDs) were all below
15%. The matrix effects (MEs) observed for all analytes in muscle,
liver, gill and gonad ranged from
16.5% to
0.73%,
4.04% to
12.4%,
7.37% to 11.3% and
13.2% to
5.35%, respectively. Detailed
method performance parameters were shown in Table S1.
2.4. Risk characterization
The health risk characterization was evaluated only for the fish
muscle in view, since muscle is the main tissue consumed by local
inhabitants. Based on “a worst-case scenario”, the maximum con-
centrations of EDCs detected in fish muscles (shown in Table 1)
were used to calculate the risks to local residents’ health after
consuming fish.
Estrogenic activity is normally calculated in terms of estradiol
464 X. Zhou et al. / Environmental Pollution 244 (2019) 462e468

Table 1
Concentrations and detection frequencies of investigated compounds in muscle, liver, gill and gonad tissues of three fish species.

Analyte Muscle Liver Gill Gonad

P. Pekinensis C. Carpio S. Chuatsi P. Pekinensis C. Carpio S. Chuatsi P. Pekinensis C. Carpio S. Chuatsi P. Pekinensis C. Carpio S. Chuatsi
(n¼21) (n ¼ 13) (n ¼ 13) (n¼21) (n ¼ 13) (n ¼ 13) (n¼21) (n ¼ 13) (n ¼ 13) (n¼21) (n ¼ 13) (n ¼ 13)

4-n-NP Range ND-2.07 ND- ND- < MQL ND-33.5 ND-148 ND-20.1 ND-22.4 ND-29.7 ND-6.63 ND-20.8 ND-10.2 ND-7.71
(ng/g) < MQL
Mean 0.17 <MQL <MQL 1.72 11.8 3.40 2.74 2.45 0.51 1.86 0.42 0.68
(ng/g)
DF (%) 9.5 7.7 7.7 9.5 15 39 29 15 7.7 14 7.7 15

BPA Range ND-3.51 0.58e1.96 1.24e4.07 ND-48.4 ND-61.9 ND-24.9 ND-48.3 ND-29.2 ND-20.0 ND-1379 ND-33.4 ND-20.7
(ng/g)
Mean 0.34* 1.15* 2.03* 4.74 13.0 5.52 9.80 9.55 5.74 13.0 12.5 5.93
(ng/g)
DF (%) 24 100 100 19 54 54 62 62 54 67 77 85

E1 Range ND-6.98 ND-4.39 ND ND-80.0 ND-55.5 ND-308 ND-37.5 ND ND ND-19.4 ND ND

(ng/g)
Mean 0.33* 1.04* ND 6.45 6.80 23.7 1.79 ND ND 1.38 ND ND
(ng/g)
DF (%) 4.8 31 ND 9.5 15 7.7 4.8 ND ND 9.5 ND ND

DES Range ND-13.0 ND-15.0 ND-15.6 ND-14.2 ND-8.12 ND ND-4.79 ND ND ND-17.1 ND ND-2.90
(ng/g)
Mean 10.9 14.6 10.1 0.79* 0.78* 0.11 0.23 ND ND 0.82 ND 0.23
(ng/g)
DF (%) 91 92 69 19 7.7 ND 4.8 ND ND 4.8 ND 7.7

EE2 Range ND ND ND ND ND ND-27.3 ND ND-5.44 ND ND ND ND

(ng/g)
Mean ND ND ND ND ND 2.10 ND 0.42 ND ND ND ND
(ng/g)
DF (%) ND ND ND ND ND 7.7 ND 7.7 ND ND ND ND

*: p value < 0.05 when compared individual EDC concentrations between different fish species by ANOVA.
ND: not detected.
DF: detection frequency.
<MQL: the concentration below the MQL.

equivalent quantity (EEQ) with a known estradiol equivalency
factor (EEF) according to (De Voogt and Van Hattum, 2003):
X thoroughly, Monte-Carlo simulation was performed to calculate the
EEQi ¼ Ci  EEFi ; and EEQt ¼ EEQi
where Ci (ng/g) is the concentration of the compound i, EEQt (ng/g)
is the total EEQ, and the EEFi is defined as the median effective
concentration (EC50) of compound i relative to the EC50 of 17b-
estradiol (E2). Literature EEF values of targeted EDCs in this study
were shown in Table 4.
Estimated daily intake (EDI) of EDC from fish consumption was
estimated by the following equation:
C  DIR
EDIi ¼ i
BW
where EDIi (mg/BWkg/day) is the EDI of the compound i, Ci (ng/g) is
the concentration of the compound i, DIR (g/day) is the daily
ingestion rate of fish muscle for local residents, and BW (BWkg) is
the body weight for consumers. In this work, DIR and BW for res-
idents in Hunan Province, China were 0.060 ± 0.046 kg/day and
58.7 ± 12.0 BWkg, respectively (Jia et al., 2018).
2.5. Statistical analysis
Concentrations of targeted EDCs below the MDLs were not
counted when calculating the detection frequency. The data below
the MQLs were set to zero for statistical analysis (Lu et al., 2017).
One-way analysis of variance (ANOVA) was used to estimate sig-
nificant differences (p < 0.05) of individual EDC among species and
tissues by Excel (Microsoft, 2016; Washington, USA). The Pearson
correlation analysis was introduced to assess the correlations
between fish sizes and EDC concentrations by SPSS software (IBM,
USA). To evaluate the human health risks by fish consumption
(1) uncertainty distributions by Oracle Crystal Ball v.11.1.41 software
(Oracle, USA).
3. Results and discussion
3.1. Comparison of EDC concentrations in wild freshwater fish
species
The concentrations of EDCs (4-n-NP, BPA, DES, E1 and EE2) in
the three fish species collected from Xiangjiang River were sum-
(2)
marized in Table 1. 4-n-NP, BPA, DES and E1 were detected in all
three species, while EE2 was only detected in C. Carpio and
S. Chuatsi.
Based on the results of the ANOVA, the significant variations of
BPA concentrations in the muscles were observed among the three
species (p < 0.05) and ranked in the order of
P. Pekinensis < C. Carpio < S. Chuatsi. Also, E1 was not detected in
muscles of S. Chuatsi, and more E1 was accumulated in that of
omnivorous C. Carpio (1.04 ng/g) rather than in that of herbivorous
P. Pekinensis (0.33 ng/g) (p < 0.05). In liver samples, the significant
variations of DES concentrations were observed between the
C. Carpio and P. Pekinensis (p < 0.05). Omnivorous C. Carpio accu-
mulated the similar contents of DES to herbivorous P. Pekinensis.
Considering that all of P. Pekinensis, C. Carpio and S. Chuatsi are
demersal fish, the reasons for the significant variations were likely
the feeding behaviors of fish species and the bioaccumulation and
biomagnification characteristics of EDCs.
The distribution characteristics of EDCs in fishes were
 X. Zhou et al. / Environmental Pollution 244 (2019) 462e468 465

Table 2
Comparison of phenols and estrogens concentrations in fish in this study with other areas in the world.

Location Species Tissues Concentration (ng/g) Reference

4-n- BPA E1 DES EE2

NP

Xiangjiang River (China) Parabramis Pekinensis, Cyprinus Carpio, and Muscle ND- ND-3.51 ND-6.98 ND- ND This study
Siniperca chuatsi 2.07 15.6
Liver ND- ND-61.9 ND-308 ND- ND-27.3
148 14.2
Gill ND- ND-48.2 ND-37.5 ND- ND-5.44
29.7 4.79
Gonad ND- ND-138 ND-19.4 ND- ND
20.8 17.1
Pearl River Estuary (China) Mugil cephalus Muscle NA 0.19e1.27 NA NA NA (Diao et al., 2017)
(dw)
Parabramis pekinensis 0.43e4.51
(dw)
Loma Lake (China) Grass Carp and Lateolabrax japonicus Muscle NA 7.56 (dw) NA 0.11 1.18 (dw) (Dan et al., 2017)
(dw)
northern coast of Sicily (Italy) Red Mullet Muscle NA 46.7e58.9 NA NA NA (Errico et al., 2017)
liver 35.0e77.6
Panlong river (China) Crucian carp and carp Muscle NA 1.9e69 NA NA NA (Wang et al., 2016)
Liver /
Gill 23
Basque Coast (Spain) Grey mullet Muscle NA 20e28 NA ND NA (Ros et al., 2016)
Liver 47e97 ND
Brain 31e46 7e8
Taihu Lake (China) Fish Muscle NA 37.3e475 4.91e364 NA 21.3e417 (Wang et al., 2015)
(dw) (dw) (dw)
Yundang Lagoon (China) Black seabream Whole NA 177 2.21 3.65 3.03 (Zhang et al., 2011)
Yellow fin seabream body 93.0 < LOD 2.85 2.71
Tilapia 54.2 < LOD 3.15 < LOD
Pearl River Delta (China) Carp Bile NA 70e1020 NA NA NA (Yang et al., 2014)
Rhone River (France) Barbel Muscle NA 3.2 NA NA NA
ge et al., 2012)
(Mie
Common bream 19.8
White bream 9.6
Chub 18.6
Anzali Wetland (Iran) Cyprinus carpino Muscle NA 1.58 ± 0.26 NA NA NA (Mortazavi et al.,
Liver 2.15 ± 0.19 2013)
Dutch water systems Bream and flounder Muscle NA <7 (dw) NA NA NA (Belfroid et al.,
(Netherlands) Liver 17 (dw) 2002)
Dianchi Lake (China) Crucian carp and carp Muscle NA 38.7 3.6 NA 3.9 (Liu et al., 2011)
Liver 107 10.7 18.7
Gill 37.5 3.5 3.3

NA: Not analyzed.

ND: Not detected.
< LOD: the concentration below the limit of detection (LOD).

Table 3
Pearson correlation coefficients between the targeted compounds and fish sizes.

Species Analyte Weight Length

Muscle Liver Gill Gonad Muscle Liver Gill Gonad

P. Pekinensis 4-n-NP
0.166 0.21
0.28 0.16 0.081 0.23
0.27 0.21
BPA
0.07 0.65**
0.27 0.03
0.22 0.36
0.20 0.06
E1 /
0.17 / 0.06 0.05
0.26 0.05 0.18
DES
0.02 0.74** 0.24
0.16 0.22 0.49** 0.41
0.21
EE2 / / / / / / / /

C. Carpio 4-n-NP
0.16 0.34
0.30
0.26
0.24
0.04
0.25
0.26
BPA 0.08 0.16 0.30 0.18 0.14 0.31 0.39 0.23
E1 0.22 0.20 / / 0.36 0.37 / /
DES
0.20
0.14 / /
0.46
0.14 / /
EE2 / /
0.07 / / /
0.04 /

S. Chuatsi 4-n-NP
0.31
0.37
0.19 0.44
0.23
0.29
0.34 0.50
BPA 0.38
0.15 0.53 0.07
0.35
0.10 0.48 0.12
E1 / 0.16 / / /
0.18 / /
DES
0.08 0.10 /
0.10
0.09 0.16 /
0.03
EE2 / 0.30 / / / 0.29 / /

**: significant at 0.01 level.

466 X. Zhou et al. / Environmental Pollution 244 (2019) 462e468
Table 4
Estrogenic activity of EDCs measured in fish samples from the Xiangjiang River.
Analyte EEFi EEQ (ng/g)
P. Pekinensis
a
4-n-NP 1.80E-07 3.73E-07
a
BPA 6.00E-04 2.11E-03
E1 0.69 b 4.82
DES 1.75 c 22.8
EE2 1.16 c / /
EEQt 27.6
a
Values were obtained from reference (Salgueiro-Gonza lez et al., 2015).
b
Values were obtained from reference (Tan et al., 2018).
c
Values were obtained from reference (Dan et al., 2017).
influenced by several physiological and ecological attributes, such
as habitat, feeding behavior and growth rate (Jia et al., 2017; Liu
et al., 2011). As for the gill and gonad samples, there was no sig-
nificant difference of EDC concentrations among different fish
species, suggesting that feeding behavior might not be a principal
factor for the bioaccumulation of EDCs in the gill and gonad.
3.2. Distributions in fish tissues and exposure mode of EDCs
The concentrations of each EDC in the muscle, liver, gill and
gonad of all the 47 fish samples are shown in Table 1. 4-n-NP, BPA,
DES and E1 were detected in all four tissues, and EE2 was only
determined in one liver and one gill.
4-n-NP was primarily detected in the liver samples and dis-
played the highest average concentration of 4.98 ng/g among four
tissues, implying that the basic metabolism in the liver was crucial
for accumulation, biotransformation and excretion of EDCs (Wang
et al., 2016). To the best of our knowledge, no previous study re-
ported the occurrence of 4-n-NP in fish samples. The fact that 4-n-
NP was found in 8.5%e19% of four tissue samples indicated its
potential bioaccumulation ability in fish, making it a candidate for a
more detailed environmental risk assessment. Several studies have
reported that the BPA concentrations in the livers were greater than
that in gills and muscles (Errico et al., 2017; Liu et al., 2011). A
similar result was also found in the present study. At the same time,
the highest average concentration and highest detection frequency
of BPA were detected in the gonads (ND-138 ng/g), which was much
higher than that in the liver (ND-61.9 ng/g). To the best of our
knowledge, no previous study has compared the levels of phenols
and estrogens in the gonads with other tissues (shown in Table 2).
This result implied that BPA could exert a negative health impact on
the reproductive function of fish directly (Liu et al., 2011). In com-
parison with previous observations, the BPA concentrations in fish
species from Xiangjiang River (ND-138 ng/g) in this study were
comparable to those from Yundang Lagoon, China (54.2e177 ng/g)
(Zhang et al., 2011) and were considerably higher than those from
the Anzali Wetland, Iran (Mortazavi et al., 2013) and the Basque
Coast of Spain (20e97 ng/g) (Ros et al., 2016).
For natural estrogen E1, the concentrations and detection fre-
quencies were also higher in the livers (ND-308 ng/g) than in other
tissues, which presented higher hepatic accumulation. For syn-
thetic estrogens, the presence of DES was found in 87% of muscle
samples and the average concentration in the muscles (11.1 ng/g)
was higher than that in other tissues. Muscle is the main tissue in
fish consumption for local inhabitants. The DES concentrations in
muscles in this study were considerably higher than those in Grass
Carp from Luoma Lake (0.11 ng/g) (Dan et al., 2017), which would
have a potentially pharmacological effect on the human health.
Among three estrogens, the detection frequency of synthetic es-
trogen EE2 was the lowest, which was only determined in one liver
Contribution (%)
C. Carpio S. Chuatsi P. Pekinensis C. Carpio S. Chuatsi
/ / / / /
1.18E-03 2.44E-03 0.01 / 0.01
3.03 / 17.5 10.3 /
26.3 27.3 82.5 89.7 99.9
/ / / /
29.3 27.3
and one gill sample at concentrations of 27.3 ng/g and 5.44 ng/g,
respectively. The low concentrations of estrogens in the fish sam-
ples that were close to or below the limit of detection were nearly
undetectable by an analytical instrument. The measurement of
estrogens has been rarely reported in fish around the word. The E1
concentrations (ND-308 ng/g) in fish species from Xiangjiang River
in present study were in the same range as in Taihu Lake, China
(4.91e364 ng/g dw) (Wang et al., 2015) and were 3e10 times higher
than those in Dianchi Lake, China (ND-3.6 ng/g) (Liu et al., 2011).
The concentrations of DES and EE2 in fish from Loma Lake, China
(0.11 ng/g and 1.18 ng/g, respectively) (Dan et al., 2017) were lower
than those in this study (ND-17.1 ng/g and ND-27.3 ng/g, respec-
tively). These differences may be attributed to the habitats, body
shapes, dietary sources and feeding behaviors of the fish.
Except for DES, the concentrations of phenols and estrogens in
organs (especially in the liver) were significantly higher than those
in the muscle (p < 0.05), which indicated that the muscle has
weaker bioaccumulation capacity for most estrogens and phenols.
In general, a liver/muscle concentration ratio >1 demonstrates the
chronic exposure to EDCs; whereas a liver/muscle concentration
ratio <1 demonstrates a short-term exposure (Errico et al., 2017). In
the present study, the mean liver/muscle concentration ratios of 4-
n-NP, BPA, E1 and EE2 were greater than 1, confirming a prolonged
contamination of these EDCs to the aquatic organism, which might
have a possible detrimental effect on protein and lipid metabolism
of the aquatic organism. In contrast, the accumulations of DES were
greater in the muscles than in the livers. Even if the fish only had a
short-term exposure to DES, the average concentration of DES in
muscle (11.1 ng/g) was considerably higher than that in the muscle
of fish from Luoma Lake, China (0.11 ng/g) (Dan et al., 2017). Since
1981, the European Union has prohibited DES from being added to
animal feed as a growth promoter (Qiao et al., 2016). However, the
high concentrations of DES in fish muscles reflected that it was still
illegally used in animal feeding operations. Therefore, it was
necessary to strengthen the supervision of hormone drugs in
animal-derived food. In addition, the gill, which was the first organ
to be in contact with water and suspended sediment particles in
aquatic environment, would be helpful to identify if the exposure
mode of the contaminants to the fish was through water exposure
(Jia et al., 2016). The concentrations of all five targeted EDCs in the
gills were not the highest, revealing that water exposure was not
the major exposure mode of these five EDCs.
3.3. Correlation analysis
The relationships between the analyzed EDC concentrations in
tissues (muscle, liver, gill and gonads) and the fish sizes (length and
weight) of three fish species were determined by Pearson corre-
lation analysis: the higher the correlation coefficient was, the
greater the strength of the relationship between the two sets of
 X. Zhou et al. / Environmental Pollution 244 (2019) 462e468 467

data was. As shown in Table 3, the correlations between fish sizes

and EDC concentrations among different fish species were
different.
In P. Pekinensis, DES concentrations in the livers showed positive
correlations with both weights(r ¼ 0.74) and lengths (r ¼ 0.49) at
significance p < 0.01. The BPA concentrations in the livers were
positively associated with weights (r ¼ 0.65) at the p < 0.01 level.
Because liver was a major site for accumulation and excretion of
contaminants, the correlations indicated that bioaccumulations of
DES and BPA increased with the growth of P. Pekinensis (Moon et al.,
1985). As for C. Carpio and S. Chuatsi, there was no significant cor-
relation between EDCs concentrations and fish sizes. In view of a
long-term exposure of fish to EDCs, this lack of correlation might be
due to that there was a balance between uptake rate and elimina-
tion rate of EDCs in fish.

3.4. Health risk assessment

Humans are not exposed to only one pollutant in their daily

lives, and the mixture of EDCs should be considered when assessing
the human health risk from the intake of these EDCs in fish
(Salgueiro-Gonz alez et al., 2013; Sebastian et al., 2013). The EEQs of
targeted EDCs and their contributions to the EEQt were shown in
Table 4.
The EEQt of analyzed compounds in muscles of P. Pekinensis,
C. Carpio and S. Chuatsi were 27.6 ng/g, 29.3 ng/g and 27.3 ng/g,
respectively. The EEQt in the C. Carpio (omnivorous) was slightly
higher than that in P. Pekinensis (herbivorous) and S. Chuatsi
(carnivorous). The main contributor to the estrogenic potential was
DES (EEFDES ¼ 1.75, 22.8 ng/g < EEQDES < 27.3 ng/g), which accounted
for more than 82.5% of the EEQt. E1 was detected in only a small
number of samples, but it was the second most relevant compound
in terms of estrogenic activity because of its relatively higher EEF
(0.69). The EEQs of BPA ranged from 1.18E-03 to 2.44E-03 ng/g.
Although BPA was one of the predominant compounds in fish
muscle samples, with maximum concentrations of more than
1.96 ng/g, it was the third contributor in terms of estrogenic activity
due to the lower EEF (6.00E-04). The contribution of 4-n-NP was
minimal. These findings implied that although estrogens have low
detection frequencies and low concentratios in the environment,
their estrogenic effects were higher than those of phenols. Therefore,
the estrogens’ impacts on human health should not be ignored.
The potential risk to local residents’ health by consuming the
three fish species from Xiangjiang River near Changsha city was
assessed through the indices of EDIs. The total EDIs of three fish
species were compared with the acceptable daily intake (ADI) of E2
to evaluate the possible human health risk. ADI of E2is 0.05 mg/
BWkg/day, according to the Joint FAO/WHO Expert Committee on
Food Additives (Library, 1974). For comparing the total EDIs with
ADI of E2, the concentration of individual EDC was transformed into
the equivalent concentration of E2, which was estimated based on
the EEFs for 4-n-NP, BPA, E1, DES, EE2 and E2 (shown in Table 4)
(Caldwell et al., 2010). The EEF for E2 is 1.0 (Tan et al., 2018).
The Monte-Carlo simulation was employed to conduct the un-
certainty of total EDIs through corresponding probabilistic mod-
elings, which were generated through 10,000 iterations. The
probabilistic distributions and descriptive statistical values of total Fig. 1. Monte-Carlo simulation results for total EDI (mg/BWkg/day) to local inhabitants:
EDIs were demonstrated in Fig. 1. As shown in Fig. 1(a), the 5th (a) P. Pekinensis, (b) C. Carpio and (c) S. Chuatsi.
percentile, median, mean and 95th percentile values for
P. Pekinensis were 0.0070, 0.0226, 0.0291 and 0.0728 mg/BWkg/day,
respectively. The first three values for the total EDI were lower than 4. Conclusions
0.05 mg/BWkg/day. However, the 95th percentile value was higher
than the limit. Moreover, the 95th percentile values for C. Carpio Among five targeted EDCs of 4-n-NP, BPA, E1, DES and EE2, the
(0.0792) and S. Chuatsi (0.0711) were also greater than 0.05 mg/ first four were detected in all of the three fish species, while EE2
BWkg/day. was only detected in C. Carpio and S. Chuatsi. Carnivorous S. Chuatsi
468 X. Zhou et al. / Environmental Pollution 244 (2019) 462e468
or omnivorous C. Carpio accumulated the higher content of BPA and
E1 than herbivorous P. Pekinensis in muscle. Moreover, the con-
centrations of steroids and phenols in the muscle, liver, gill and
gonadwere variable. The liver presented a higher affinity for the
accumulation of 4-n-NP and E1 than other tissues. BPA and DES
primarily accumulated in the gonads and muscles, respectively.
According to the liver/muscle concentration ratio, the aquatic or-
ganisms were chronically exposed to 4-n-NP, BPA, E1 and EE2. In
addition, bioaccumulation of DES and BPA increased with the
growth of P. Pekinensis on the basis of the results of Pearson cor-
relation analysis. Regarding C. Carpio and S. Chuatsi, fish sizes had
little effect on the bioaccumulation of EDCs. Taking co-exposure
into account, the estrogenic effects of estrogens were higher than
those of phenols. According to the results of the Monte-Carlo
simulation, the 95th percentile values of total EDIs by consuming
the three freshwater fish species from Xiangjiang River were higher
than the ADI of E2.
Declarations of interest
None.
Acknowledgments
This work was supported by the Special Fund for Agro-Scientific
Research in the Public Interest of China (Grant No. 201503108) and
the Science and Technology Project of Hunan Province (Grant No.
2017WK2091).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.envpol.2018.10.026.
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