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Analysis of estrogenic activity in environmental waters in Rio de Janeiro state (Brazil) using the yeast estrogen screen
Ecotoxicology and Environmental Safety 120 (2015) 41–47
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Analysis of estrogenic activity in environmental waters in Rio de Janeiro state (Brazil) using the yeast estrogen screen Amanda Cristina Vieira Dias a, Frederico Wegenast Gomes a, Daniele Maia Bila b,n, Geraldo Lippel Sant’Anna Jra, Marcia Dezotti a a b
Chemical Engineering Program – COPPE, Federal University of Rio de Janeiro, P.O. Box 68502, 21941-972 Rio de Janeiro, RJ, Brazil Department of Environmental and Sanitary Engineering, State University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil
art ic l e i nf o
a b s t r a c t
Article history: Received 11 November 2014 Received in revised form 7 May 2015 Accepted 10 May 2015 Available online 27 May 2015
The estrogenicity of waters collected from an important hydrological system in Brazil (Paraiba do Sul and Guandu Rivers) was assessed using the yeast estrogen screen (YES) assay. Sampling was performed in rivers and at the outlets of conventional water treatment plants (WTP). The removal of estrogenic activity by ozonation and chlorination after conventional water treatment (clarification and sand filtration) was investigated employing samples of the Guandu River spiked with estrogens and bisphenol A (BPA). The results revealed a preoccupying incidence of estrogenic activity at levels higher than 1 ng L 1 along some points of the rivers. Another matter of concern was the number of samples from WTPs presenting estrogenicity surpassing 1 ng L 1. The oxidation techniques (ozonation and chlorination) were effective for the removal of estrogenic activity and the combination of both techniques led to good results using less amounts of oxidants. & 2015 Elsevier Inc. All rights reserved.
Keywords: Endocrine disrupting chemicals Estrogens YES assay River water quality
1. Introduction Since the 1990s, when the occurrence of endocrine disrupting chemicals (EDCs) influencing the sexual development of fish in English rivers was reported (Purdom et al., 1994), the presence of compounds that potentially interfere with the endocrine system of humans and wildlife has become a major concern worldwide (Streck, 2009). Exposure to EDCs has been associated with damage to the male and female reproductive systems, developmental problems, a decline in the ratio between male and female individuals, changes in the thyroid gland and neurobehavioral effects (Koifman et al., 2002; Pasqualotto et al., 2004; Coleman et al., 2005; James, 2006; Paris et al., 2006). According to Hotchkiss et al. (2008), new concerns have been expressed by scientists regarding the potential role of EDCs in precocious puberty in girls, obesity and type II diabetes in the United States and other countries as well as in relation to complex endocrine alterations induced by mixtures of a variety of potent human and veterinary n Correspondence to: State University of Rio de Janeiro (UERJ), Department of Sanitary and Environmental Engineering, Rua Sao Francisco Xavier 524, 5029-F, CEP 20550-900 Rio de Janeiro, RJ, Brazil. E-mail addresses: [email protected] (A.C.V. Dias), [email protected] (F.W. Gomes), [email protected] (D.M. Bila), [email protected] (G.L. Sant’Anna Jr), [email protected] (M. Dezotti).
http://dx.doi.org/10.1016/j.ecoenv.2015.05.013 0147-6513/& 2015 Elsevier Inc. All rights reserved.
pharmaceutical products, personal care products, nutraceuticals and phytosterols present in the environment. Endocrine disruptors, such as estrogenic chemicals, are introduced into the aquatic environment by human activity, both domestic and industrial. In developed countries, effluents from municipal wastewater treatment plants (WWTPs) are the main source of estrogenic chemicals in natural waters, because these substances are not completely removed in conventional treatment processes (D’Ascenzo et al., 2003; Auriol et al., 2006). In developing countries, like Brazil, the problem seems to be more acute, since wastewaters without previous treatment are commonly released into the environment and this is certainly the major source of estrogenic compounds in surface waters. The Brazilian population (203 million inhabitants) is concentrated in urban areas. The resulting intensive soil occupation coupled with inadequate infrastructure for sewage collection and treatment has a negative effect on the quality of natural waters. Despite the fact that Brazil has abundant sources of fresh water and high-capacity water treatment plants (WTPs), such as that located in the metropolitan area of Rio de Janeiro (the largest WTP in the world, with the capacity to treat continuously 43 m3/s of raw water), few studies on the occurrence of estrogenic compounds in local natural water sources could be found in the literature (Ternes et al., 1999; Araújo 2005; Ghiselli, 2006; Raimundo CCM, 2007; Gerolin ERR, 2008; Reis Filho, 2008;
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Kuster et al., 2009). Among the cited authors, only Kuster et al. (2009) evaluated water samples collected from the main water supply sources of Rio de Janeiro State. Reis Filho (2008) discussed the biological effects associated with the presence of estrogenic compounds in natural waters in Sao Paulo State. The presence of estrogenic compounds in river samples was observed in both studies: in the former the occurrence of phytoestrogens, both free and conjugated estriol (E3), and conjugated 17β-estradiol (E2) in natural samples was reported and in the latter strong evidence of endocrine disruption in male fish due to the presence of estrogenic compounds and the presence of 17α-ethinylestradiol (EE2) in some river samples were verified. It is widely known that in chemical analysis the interactions in mixtures are not taken into consideration and, consequently, accessing the biological effects of the water matrix will require the use of other types of assays (Routledge and Sumpter, 1996). The yeast estrogen screen (YES) is one of the most common in vitro bioassays used to provide initial evidence of estrogenic contaminants in ecosystems. This is a robust, rapid and sensitive tool for assessing estrogenic activity in environmental samples at moderate cost (Beck et al., 2006). The YES assay is used to evaluated the estrogenic activity in waters and wastewaters (Brix et al., 2010; Li et al., 2014). The lack of information regarding endocrine disruption activity caused by the presence of estrogenic compounds in Brazilian natural waters and treated water for public supply motivated us to carry out this study. The YES assay was used to assess the estrogenic activity in natural waters in Rio de Janeiro State (Paraíba do Sul-Guandu River system). The location of the aquatic environment investigated is shown in Supplementary information, Fig. S1 Samples were collected in six sampling campaigns performed between March 2007 and December 2008. The determination of the estrogenicity of both raw and treated water samples was also included in the experimental work. Moreover, the estrogenic activity in water samples collected from the Guandu River was monitored and its removal during oxidative treatment by ozonation and chlorination was evaluated in bench-scale experiments performed in our laboratory. The aim of this research was to contribute to the determination of the degree of pollution in important aquatic environments in Rio de Janeiro State and evaluate the potential of ozonation and chlorination to reduce the estrogenic activity.
2. Materials and methods 2.1. Reagents Stock solutions of 17β-estradiol (E2) and 17α-ethinylestradiol
(EE2) (98% purity, Sigma-Aldrich) were prepared at 100 mg L 1 in acetone and stored at 4 °C. All medium constituents were obtained from Sigma-Aldrich except biotin, which was supplied by Merck. Purified water was obtained from a Milli-Q Biocell system (Millipore). Chlorophenol red-β-D-galactopyranoside (CPRG) was supplied by Roche Diagnostics GmbH. For the sample extraction, hexane, methanol and acetone (HPLC and spectrophotometric grade solvents) were supplied by Tedia Brazil and ethanol (HPLC grade) and BPA were obtained from Merck. 2.2. Area of study The state of Rio de Janeiro, whose capital (the city of Rio de Janeiro) was chosen to host the 2016 Olympics Games, is located in southeastern Brazil and has around 15.2 million inhabitants (IBGE, 2010a). Approximately 6.3 million live in the central city area of Rio de Janeiro and 12.4 million in the wider metropolitan area. Other important towns in this state, which lie far from the capital, are Campos dos Goytacazes, Resende and Volta Redonda. The Guandu and Paraiba do Sul Rivers are the most important sources of water in the state of Rio de Janeiro, since together they supply more than 12 million people, including 85% of the metropolitan area residents. The Paraiba do Sul & Guandu system supplies electric power and water for several cities. A flow rate of 160 m3 s 1 is diverted from the Paraiba do Sul River to the Guangu River to assure water supply for the city of Rio de Janeiro. All sampling locations are shown in Supplementary information, Fig. S1, highlighted by black circles which in the case of sampling locations 3 and 5 represent more than one sampling site. Sampling dates, site codes and the respective GPS coordinates are listed in Table 1. The sampling sites 1, 2 and 3 are located in medium-sized cities and represent the most populated and industrialized region along the Paraiba do Sul River in the studied area. Site 1 is the closest to the boundary between the states of Sao Paulo and Rio de Janeiro and represents the quality of the Paraiba do Sul River as it flows into Rio de Janeiro State. At site 3, samples of water from the Paraiba do Sul River treated in a WTP (Tw3 samples) were also collected during all sampling campaigns carried out in 2008. Sampling site 4 is located in the north of the state, along the estuary of the Paraiba do Sul River, in the town of Campos dos Goytacazes. In this region agriculture and agro-industry are important economic activities. Sampling site 5 is located in Nova Iguacu where water from the Guandu River is driven to the water treatment plant and subsequently supplied to Rio de Janeiro metropolitan area. At this location, treated and raw water samples were collected. The water
Table 1 Description of water sampling sites in selected areas of Rio de Janeiro State. CodeFig. 1
Location (inhab.)a
1 2 3
Resende (130,035) Barra Mansa (176,899) Volta Redonda (261,404)
4 5
C. Goytacazesb (431,839) Nova Iguaçu
a b c
Samplecode
Source
PS1 PS2 PS3 Tw3c PS4 G1 G2
Paraiba do Sul Paraiba do Sul Paraiba do Sul Tap water Paraiba do Sul Guandu river Guandu river
Tw5c
Tap water
GPS coordinates
river river river river
o
o
22 .27.94’S44 .26.90’W 22o.31.50’S 44o.11.33’W 22o.30.05’S44o.05.42’W 22o.30.11’S44o.05.44’W 21o.44.47’S 41o.20.16’W 22o.49.28’S43o.37.31’W 22o.48.70’S43o.37.71’W 22o.49.76’S43o.36.99’W
Sampling data and campaigns 2007
2008
Mar. Apr. Mar. Apr. Mar. Apr. Oct.
Jan. Jan. Jan. Jan.
Mar. Apr Mar. Apr. Sep.
Jul. Jul. Jul. Jul.
Sep. Sep. Sep. Sep.
Jan. Jun. Jul. AugSep. Oct. Nov. Dec. Mar. Apr. Sep. Jan. Aug. Nov
IBGE (2009). Campos dos Goytacazes. Treated water from different WTP (primary treatment processes: coagulation and flocculation, sand filtration and chlorination).
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samples from the Guandu River were initially collected along its left margin (G1 samples). However, the sampling location was transferred to a nearby bridge, close to the Guandu WTP inlet (G2 samples), in order to evaluate the estrogenic activity at the point of greatest clearance (far from the points of contamination along the river margins). Samples of the water treated at the Guandu Water Treatment Plant were also collected at the exit of the plant (Tw5) and the estrogenic activity was determined. 2.3. Estrogenicity removal by ozonation and chlorination All samples were filtered and concentrated by solid phase extraction before evaluation of the estrogenic activity and data were expressed as 17β-estradiol (E2) equivalents (E-EQ). To simulate the water treatment performed in local WTPs, coagulation was conducted using FeCl3 followed by sedimentation in a jar-test apparatus as follows: ferric chloride dose of 35 mg L 1, strong agitation for 5 min, mild agitation for 20 min and sedimentation for 30 min. The supernatant was then filtered through a sand filter (bed height of 25 cm) containing particles of sizes ranging from 0.4 to 0.9 mm. The water sample was then filtered through 1.2 μm glass fiber filters and chlorinated (NaClO) and/or ozonated. Ozonation was carried out in a system comprised of a glass contact column (1 L), an ozone generator (Unitek – model UTK) and an ozone analyzer (IN USA, ASX-Mod H1) for the gas phase. Ozone doses in the range of 0.5–3.5 mg O3 L 1 were applied. Chlorination was conducted using chlorine doses of 1.5 and 3.2 mg Cl2 L 1 All experiments were performed at 247 2 °C and pH was in the range of 6–7. Since the estrogenic activity in the Guandu River water was low, to investigate the potential of lab-scale chlorination and ozonation to remove estrogenic activity, samples were spiked with E2, EE2 and BFA to give the following concentrations: E2 (2.5 and 5 ng L 1), EE2 (2.5 and 5 ng L 1) and BFA (0, 500 and 1000 ng L 1). 2.4. Water quality: analytical determinations All aqueous samples (5 L) were collected in acetone-rinsed amber glass bottles and returned to the laboratory where they were immediately characterized by measuring the dissolved organic carbon (DOC), turbidity, pH, conductivity and N-NH3, according to APHA (2005). 2.5. Sample preparation for the estrogenic activity assay All samples were filtered through 1.2 μm glass fiber filters (Millipore) immediately after collection. Aliquots (1 L) of the samples collected in 2007 and in January 2008 were also filtered through 0.45 μm cellulose acetate filters (Sartorius, Göttingen, Germany) and 0.22 μm glass fiber filters (MFS, USA) in parallel, and the estrogenic activity of each filtrate was determined. The filtered samples were acidified to pH 3 and stored at 4 °C until extraction, which was performed within 48 h after sampling. After the filtration, all samples were concentrated by solid phase extraction (SPE) before the YES assay. SPE was performed using 500 mg and 2 g Bond Elut C18 cartridges (Varian) for samples filtered through both 0.22 μm and 0.45 μm filters and for samples filtered through a 1.2 μm filter, respectively. Immediately before use the cartridges were conditioned with hexane, acetone and methanol and washed with purified water at pH 3. The prefiltered samples were forced under vacuum through the cartridge at a flow-rate of approximately 10 mL min 1. The cartridges were kept under vacuum aspiration for 30 min after the extraction had finished. The elution of analytes from the cartridges was performed using 4 mL of acetone.
43
The solvent was evaporated under a gentle nitrogen stream to dryness. Finally, the dry samples were reconstituted with 2 mL of ethanol and stored at 18 °C until analysis applying the YES assay. 2.6. Yeast estrogen assay The estrogenic activity of the sample extracts was determined using a recombinant reporter gene assay known as yeast estrogen screen (YES). This recombinant Saccharomyces cerevisiae cell line contains the human estrogen receptor gene linked to a receptor gene coding for β-galactosidase and was kindly provided by Prof. Sumpter, Brunel University, UK. In the presence of estrogenic chemicals, this enzyme is produced and secreted into the assay medium, where it breaks down the chromogenic substrate CPRG, and the color change is quantified. Yeast cells were cultured and the assay was carried out as described by Routledge and Sumpter (1996) with some modifications. The E2 standard solution and sample extracts were serially diluted in ethanol and 10 μL of each dilution were transferred (at least in duplicate) into a 96-well optically flat microtiter plate and allowed to evaporate until dryness. Next, 200 μL of seeded yeast medium containing CPRG were added to the wells. The plates were then sealed with autoclave tape and shaken vigorously for 2 min. After incubation for 72 h at 30 °C the absorbance was read at 540 nm (for color) and at 620 nm (for turbidity) using a plate reader (BIO-TEK EL808). In each test, ethanol and E2 (serially diluted) were used as blank solvent control and positive control, respectively. 2.7. Data analysis The estrogenic responses for the sample extract and E2 standard were plotted after being corrected for turbidity (Coleman et al., 2004). Inhibition of yeast cell growth was taken as an acute toxic effect of a tested sample and was observed a reduction in the absorbance at 620 nm, compared to the reference wells (i.e., solvent control) (Aerni et al., 2004; Beck et al., 2006). Calibration dose–response curves for E2 standard were plotted giving concentration versus estrogenic response in corrected absorbance at 540 nm. The resulting sigmoidal curves were fitted to a symmetric logistic function (Origin 6.0, Microsoft, USA). The estrogenic activity in each well containing sample extract was then calculated as E2 equivalents (E-EQ) by interpolation from the E2 standard curves (in μg L 1). These values were divided by the SPE concentration factor, resulting in the final concentration for the water sample as E-EQ (range of ng L 1). In this study, dose–response curves for E2 were obtained for a concentration range of 1.36 μg L 1 to 0.67 ng L 1 in the wells (Rudder et al., 2004; Fine et al., 2006).
3. Results and discussion 3.1. Water quality The characteristics of the water samples collected in different places are shown in Table 2. The dissolved organic carbon (DOC) was between 3 and 16.6 mg L 1 and for most samples it was lower than 7 mg L 1. Turbidity ranged from 3.4 to 25.2 NTU and the pH was close to 7 for all samples. Levels of N-NH3 were, in general, low (between 0.2 and 1.1 mg L 1) and water conductivity was below 100 mS cm 1 with the exception of one sample for which the conductivity surpassed this upper limit.
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Table 2 Water quality of samples collected in different river locations. Source
Location
Sample code
Sampling date
DOC (mg L 1)
Turbidity (NTU)
pH
Conductivity (mS cm 1)
N-NH3 (mg L 1)
PS Rivera
Resende
PS1
Barra Mansa
PS2
Volta Redonda
PS3
Campos Goytacazes
PS4
3/15/2007 4/16/2007 1/21/2008 7/23/2008 9/30/2008 3/15/2007 4/16/2007 1/21/2008 7/23/2008 9/30/2008 3/15/2007 4/16/2007 10/2/2007 1/21/2008 7/23/2008 9/30/2008 3/20/2007 4/18/2007
8.5 4.6 3.7 8.5 6.4 5.4 3.0 – 6.7 4.2 10.4 4.4 6.0
10.2 8.2 19.4 3.4 5.3 11.0 10.2 25.2 4.4 6.0 12.7 8.0 5.9 24.0 4.4 7.5 21.7 25.4
6.7 6.8 6.5 7.2 7.2 6.5 7.4 6.6 7.4 7.3 6.7 7.0 6.7 6.6 7.2 7.3 7.0 7.0
79.0 85.3 91.9 86.7 86.1 76.5 78.5 72.3 84.5 86.3 80.6 103.3 98.6 86.4 83.9 94.5 61.1 64.4
0.2 0.2 0.6 0.6 0.4 0.2 0.5 0.4 0.6 0.4 0.3 0.3 1.5 0.7 1.1 0.6 0.4 0.2
Guandu River
Nova Iguaçu
G1
3/15/2007 4/16/2007 9/27/2007 1/28/2008
6.4 16.6 5.9
16.3 8.1 4.5 17.3
6.8 6.7 6.9 6.6
77.6 84.6 96.2 86.1
0.3 0.2 2.6 0.4
Guandu River
Nova Iguaçu
G2
7/1/2008 7/9/2008 8/1/2008 8/19/2008 8/27/2008 9/17/2008 10/9/2008 10/22/2008
6.7 5.6 3.9 4.3 4.6 3.1 4.5 3.0
5.4 5.3 4.5 4.6 5.0 4.7 24.2 8.2
6.8 7.0 7.0 7.5 7.6 7.2 7.1 7.4
– – 88.7 94.8 96.9 96.2 85.6 81.8
0.4 0.5 03 – 0.8 0.4 – 1,1
a
Paraiba do Sul River.
3.2. Estrogens in Rio de Janeiro state water sources The sanitation conditions in many municipalities in the southeast region of Brazil can be described as poor. Data for 2008 reveal that, out of 1668 municipalities in this region, 1586 have sewage collection systems in operation. However, only 808 municipalities have sewage treatment facilities and treatment of sewage at a secondary level (biological) is only performed in 461 municipalities (IBGE, 2010b). Thus, in many localities sewage reaches the aquatic systems without any type of treatment or is precariously treated. The estrogenic activity results are shown in Fig. 1. The seasonal variations will have an effect on the results, particularly the E-EQ (ng/L) 15
10
03/15/07 04/16/07 01/21/08 07/23/08 09/30/08
03/15/07 04/16/07 01/21/08 07/23/08 09/30/08
03/15/07 04/16/07 10/02/07 01/21/08 07/23/08 09/30/08
03/20/07 04/18/07
03/15/07 04/16/07 09/27/07 01/28/08
07/01/08 07/09/08 08/01/08 08/19/08 08/27/08 09/17/08 10/09/08 10/22/08
5
PS1
PS2
PS3
PS4
G1
G2
Fig. 1. Estrogenic activity results: white bars relate to samples filtered through 0.45 μm membrane and black bars to samples filtered through 1.2 μm membrane.
raining periods in summer (December to March). In Fig. 1, white and black bars correspond to samples filtered through 0.45 and 1.2 μm filters, respectively. Since samples filtered through 0.22 μm and 0.45 μm membranes led to almost the same results, only data from the latter case are reported herein. Since adverse effects of estrogens have been observed at 1 ng L 1 (Routledge et al. 1998, Petrovic et al., 2002), this value was taken as a reference in this study and is represented by the dashed line in Figs. 1 and 2. The limit level of 1 ng L 1 was also considered by Matthiessen. et al. (2006) in their work with livestock farms streams. The data shown in Fig. 2 reveals that values far higher than this limit were observed in some samples from all sampling points. Several samples filtered through a 1.2 μm filter showed notably high E-EQ values. In order to obtain a more consistent view of the E-EQ results, values surpassing 1 ng L 1 and their incidence are reported in Table 3 for all sampling campaigns. Values of E-EQ lower than 1 ng L 1 were observed for samples Resende (PS1), Volta Redonda (Tw3) and C. Goytacazes (PS4) filtered through a 0.45 μm filter and also for Volta Redonda (Tw3) for samples filtered through a 1.2 μm filter. In general, the estrogenicity of the river water samples seems to increase as the Paraiba do Sul River flows through the Rio de Janeiro State and reaches maximum values at Volta Redonda (PS3). For samples collected from the Guandu River (G1 and G2) 8% of the E-EQ values were higher than 1 ng L 1. However, a matter of concern is the high incidence (25%) of water samples treated by conventional techniques (Tw5, filtered through 1.2 μm) presenting E-EQ values which surpass this limit. The high incidence (50%) observed for samples collected from C. Goytacazes (PS4) and filtered through a 1.2 μm filter should be considered with prudence, since the number of samples collected at this site was small. The presence of estrogens and progestogens in the Paraiba do
A.C.V. Dias et al. / Ecotoxicology and Environmental Safety 120 (2015) 41–47
contrast to estradiol 17-glucoronide and estriol 16-glucoronide which do not give any response in the assay. However, these glucoronides are easily converted to estriol and estradiol by enzymes produced by bacteria found in sewage and waters. Thus, these substances and the non-conjugated hormones (estriol and estradiol), which were also detected by Kuster et al. (2009), could be responsible for the estrogenic activity found in detectable and significant levels in the aquatic system investigated in the study reported herein, which receives important amounts of non-treated sewage.
E-EQ (ng.L-1) 15
10
10.3
9.7
9.0
5
0.9
RWs
O (3.5 mg.L-1 )
CFW
CW
45
3.3. Estrogenicity removal by chlorination and ozonation E-EQ (ng.L-1) 15
10
5
5.6
4.8
3.2 0.8
RWs
CW
O3 (1 mg.L-1)
CFW
0.9
0.3
O3 (3.5 mg.L-1)
O3 (2 mg.L-1)
E-EQ (ng.L-1) 15
10
5 2.3
2.7 1.3
RWs CW
0.3
0.3
O3 O3 O3 (0.5 mg.L-1) (1 mg.L-1) (2 mg.L-1)
0.4
0.2
Cl2 Cl2 (1.5 mg.L-1) (3.2 mg.L-1)
0.1
Cl2 (1.5 mg.L-1) O (0.5 mg.L-1)
Fig. 2. Estrogenic activity of Guandu River water spiked with: (a) E2, EE2 (5 ng L 1) and BPA (1000 ng L 1); (b) and (c) E2, EE2 (2.5 ng L 1) and BPA (500 ng L 1). All samples were treated by clarification, sand filtration and ozonation. RWs – River water (spiked), CW – Clarified water, CFW – Clarified and filtrated water.
Sul and Guandu Rivers has been previously detected by Kuster et al. (2009). These authors found appreciable levels of progesterone in samples collected from Volta Redonda, Barra Mansa and Guandu River. In addition, estriol, estradiol 17-glucoronide and estriol 16-glucoronide were detected in water samples collected from these locations. In the treated Guandu River water, estradiol 17-glucoronide and progesterone were also detected. Progesterone, possibly due to its chemical structure, gives an estrogenicity response in the YES assay (Wang et al., 2005) in
In previous studies carried out in our laboratory ozone was able to remove the estrogenicity associated with 17β-estradiol (Bila et al., 2007) and that related to both 17β-estradiol and 17α-ethinylestradiol (Maniero et al., 2008) in pure water. The results obtained motivated us to investigate ozonation alone or in combination with chlorination in order to remove or reduce estrogenic EDCs from a more complex matrix (river water). The Guandu River water was treated in the laboratory, as described in Section 2.3. The treatment was able to reduce turbidity to a large extent, as shown by the data in Table 4. The pH of the water samples dropped slightly after treatment and remained in the range of 6.0–6.6. Table 4 summarizes the experimental conditions used in ozonation and chlorination experiments. In the last three experiments ozonation and chlorination were applied in the water treatment. Fig. 2a shows the results obtained in assays conducted with the highest concentrations of E2 and EE2 (5 ng L 1) and BFA (1000 ng L 1). The estrogenicity of the water spiked with these substances was not removed after clarification and sand filtration. However, it was reduced to an average value of 0.9 ng L 1 E-EQ after ozonation (3.5 mgO3 L 1). Fig. 2b shows similar results for initial E2, EE2 and BFA concentrations of 2.5, 2.5 and 500 ng L 1, respectively. A pronounced estrogenicity reduction was enhanced by ozonation at the three ozone doses tested. In experiments without BFA and with E2 and EE2 concentrations of 2.5 ng L 1, ozonation and chlorination alone or combined were investigated. As shown in Fig. 2c, ozone doses of 1 and 2 mgO3 L 1 were able to reduce estrogenic activity to an average value of 0.3 ng L 1 E-EQ. Chlorination was also effective in the removal of estrogenicity using doses of 1.5 and 3.2 mgCl2 L 1. However, an interesting result was obtained combining a low ozone dose (0.5 mgO3 L 1) with a low chlorine dose (1.5 mgCl2 L 1). This condition led to the lowest residual estrogenic level in the water (0.1 ng L 1). Thus, the combination of chlorination and ozonation seems an interesting alternative to remove estrogenicity and reduce the use of oxidants. The results obtained in this study revealed the presence of
Table 3 Maximum E-EQ values and incidence of values higher than 1 ng/L. Location
Sample code Sampling date
Maximum E-EQ (ng L 1) f o 0.45 μm
Resende Barra Mansa
PS1 PS2
0.9 1.7
Volta Redonda Volta Redonda Nova Iguaçu Nova Iguaçu Nova Iguaçu C.Goytacazes
PS3 Tw3 G1 G2 Tw5 PS4
3.1 ndl 1.3 0.5 0.9 0.5
03/15/2007 09/30/200803/15/ 2007 03/15/2007 01/21/2008 03/15/2007 10/22/2008 03/15/2007 03/20/2007
Maximum E-EQ (ng L 1) f o1.2 μm
E-EQ41 ng/L incidencen (%)
0 9
2.9 4.8
20 20
17 0 25 0 0 0
16 ndl 15 2.3 10 1.3
50 0 8 13 25 50
E-EQ41 ng/L incidencen (%)
ndl ¼ non detectable levels. n
ncidence ¼ (number of samples with E-EQ 4 1 ng/L).100/(total number of samples).
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Table 4 Experiments with Guandu River water spiked with different concentrations of E2, EE2 and Bisphenol A (BFA). River water and treated water characteristics
Oxidation experiments conditions
Sampling Date
River water pH turbidity (NTU)
Treated water pH turbidity (NTU)
Ozone and/or chlorine doses and initial concentrations
09/12/2008 09/19/2008 10/22/2008 11/14/2008 12/11/2008 12/17/2008
7.0 7.2 7.4 7.5 7.4 7.5
6.2 6.0 6.2 6.3 6.6 6.4
3.5 mgO3 L 1; E2 and EE2 (5 ng L 1), BFA (1000 ng L 1) 3.5 mgO3 L 1; E2 and EE2 (2.5 ng L 1), BFA (500 ng L 1) 1 and 2 mgO3 L 1; E2 and EE2 (2.5 ng L 1), BFA (500 ng L 1) 0.5; 1 and 2 mgO3 L-1; 1.5 and 3.2 mgCl2 L-1; E2 and EE2 (2.5 ng L-1) 0.5; 1 and 2 mgO3 L 1; 1.5 and 3.2 mgCl2 L 1; E2 and EE2 (2.5 ng L 1) 0.5 and 1 mgO3 L 1; 1.5 mgCl2 L 1; E2 and EE2 (2.5 ng L 1)
4 4 11 16 18 34
0.02 0.02 0.10 0.14 2.1 2.2
estrogenic activity in the river water samples and also in samples collected downstream of the water treatment plants (WTPs). Although chlorination is a necessary and effective process in WTPs, mainly in tropical countries due to the high temperatures and the extension of the water distribution system, it should be combined with other processes to assure estrogenicity removal. The results reported herein highlight the need to improve the technology used in WTPs, chiefly in countries which have emerging and important economies, like Brazil.
agencies (CNPq and FAPERJ).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at
References 4. Conclusions The estrogenic activity of river waters (Paraiba do Sul and Gundu Rivers) is a matter of concern. For samples filtered through 1.2 μm filters, the estrogenic activity increased as the Paraiba do Sul River flowed through the Rio de Janeiro State. Maximum values of 2.9, 4.8 and 16 ng L 1 E-EQ were observed in samples collected at Resende, Barra Mansa and Volta Redonda, respectively. The percentage of samples (20–50%) collected from these sites which presented estrogenic activity was also a matter of concern. The discharge of raw and treated sewage into the river is common in cities due to deficient sanitation and treatment systems. In the case of the Guandu River, the incidence of samples presenting estrogenic activity higher than 1 ng L 1 E-EQ was 8%. However, very high maximum values (15 ng L 1 E-EQ) were observed for samples filtered through a 1.2 μm filter and a preoccupying result was the incidence of 25% of samples presenting activity higher than 1 ng L 1 after the treatment of Guandu River water by conventional techniques. The results obtained in this study demonstrate that water contamination by endocrine disrupting chemicals is occurring at several locations along the Paraiba do Sul and Guandu Rivers. Furthermore, estrogenic activity was observed in water supply systems, indicating that water treatment plants should be redesigned in order to achieve the removal of endocrine disrupting chemicals. The results for the Guandu River water treated by chemical clarification, sand filtration and ozonation and/or chlorination were promising, since ozonation was effective in reducing the estrogenicity levels of 5 and 2.5 ng L 1 E-EQ to values far below the reference value of 1 ng L 1. In addition, the combination of low ozone and chlorine doses (0.5 mgO3 L 1 and 1.5 mgCl2 L 1) promoted a considerable reduction in the estrogenic activity using relatively low doses of oxidants. In developing countries the chlorination process is already applied in WTPs but ozonation is not widely used. Thus, investments in ozonation or other processes that are effective for the removal of EDCs are required to ensure the supply of water free from these micropollutants.
Acknowledgements Authors express their gratitude to the following Brazilian
Aerni, H.-R., Kobler, B., Rutishauser, B.V., et al., 2004. Combined biological and chemical assessment of estrogenic activities in wastewater treatment plant effluents. Anal. Bioanal. Chem. 378, 688–696. APHA, AWWA, WEF, 2005. Standard Methods for the Examination of Water and Wastewater. 22th ed., New York. Araújo, J.C., 2005. Study of the Efficiency of the Treatment of Domestic Effluents of the City of Araraquara-SP in the Removal of Sexual Hormones. Dissertation (in Portuguese), Universidade de Sao Paulo, Sao Carlos. Auriol, M., Filali-Mehnassi, Y., Tyagi, R.D., Adams, C.D., Surampalli, R.Y., 2006. Endocrine disrupting compounds removal from wastewater, a new challenge. Process Biochem. 41, 525–539. Beck, I.-C., Bruhn, R., Gandrass, J., 2006. Analysis of estrogenic activity in coastal surface waters of the Baltic Sea using the yeast estrogen screen. Chemosphere 63, 1870–1878. Bila, D.M., Montalvao, A.F., Azevedo, D.A., Dezotti, M., 2007. Estrogenic activity removal of 17β-estradiol by ozonation and identification of by-products. Chemosphere 69, 736–746. Brix, R., Noguerol, T.N., Piña, B., Balaam, J., Nilsen, Aj, Tollefsen, K.E., Levy, W., Schramm, K.E., Barceló, D., 2010. Evaluation of the suitability of recombinant yeast-based estrogenicity assays as a pre-screening tool in environmental samples. Environ. Int. 36, 361–367. Coleman, H.M., Routledge, E.J., Sumpter, J.P., 2004. Rapid loss of estrogenicity of steroid estrogens by UVA photolysis and photocatalysis over an immobilized titanium dioxide catalyst. Water Res. 38, 3233–3240. Coleman, H.M., Abdullahb, M.I., Eggins, B.R., et al., 2005. Photocatalytic degradation of 17β-oestradiol, oestriol and 17α-ethynyloestradiol in water monitored using fluorescence spectroscopy. Appl. Catal. B: Environ. 55, 23–30. D’Ascenzo, G., Di Corcia, A., Gentili, A., et al., 2003. Fate of estrogen conjugates in municipal sewage transport and facilities. Sci. Total Environ. 302, 199–209. Fine, T., Leskinen, P., Isobe, T., et al., 2006. Luminescent yeast cells entrapped in hydrogels for estrogenic endocrine disrupting chemical biodetection. Biosens. Bioelectron. 21, 2263–2269. Gerolin, E.R.R., 2008. Occurrence and Removal of Endocrine Disruptor Compounds in Drinking Water at Campinas and Sumare, Sao Paulo State, Brazil. D.Sc. Thesis (in Portuguese) Universidade Estadual de Campinas (Unicamp). Ghiselli, G., 2006. Potable Water Quality in the Campinas Region: Occurrence and Determination of Endocrine Disruptors (EDs) and Pharmaceuticals and Personal Care Products (PPCPs). D.Sc. Thesis (in Portuguese) Universidade Estadual de Campinas (Unicamp). Hotchkiss, A.K., Rider, C.V., Blystone, C.R., Wilson, V.S., et al., 2008. Fifteen years after “Wingspread” – environmental endocrine disrupters and human and wildlife health: where we are today and where we need to go. Toxicol. Sci. 105, 235–259. IBGE, 2010a. Brazilian Institute of Geography and Statistics. Census 2010, Rio de Janeiro, Brazil. IBGE, 2010b. Brazilian Institute of Geography and Statistics. National Survey of Basic Sanitation (PNSB), 2008, Rio de Janeiro, Brazil. James, W.H., 2006. Offspring sex ratios at birth as markers of paternal endocrine disruptors. Environ. Res. 100, 77–85. Kuster, M., Azevedo, D.A., López de Alda, M.J., Aquino Neto, F.R., Barcelo, D., 2009. Analysis of phytoestrogens, progestogens and estrogens in environmental waters from Rio de Janeiro (Brazil). Environ. Int. 35, 997–1003. Koifman, S., Koifman, R.J., Meyer, A., 2002. Human reproductive system disturbances and pesticide exposure in Brazil. Cad Saude Publica 18, 435–445. Li, B., Cao, J., Xing, C., Wang, Z., Cui, L., 2014. Assessing estrogenic activity and eproductive toxicity of organic extracts in WWTP effluents. Environ. Toxicol.
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Portuguese), Universidade Estadual de Campinas (Unicamp). Reis Filho, R.W., 2008. Estrogen hormones in Monjolinho river, Sao Carlos-SP: an assessment of environmental endocrine disruptors. D.Sc. Thesis (in Portuguese) Universidade de Sao Paulo, Sao Carlos, SP, Brazil. Routledge, E.J., Sumpter, J.P., 1996. Estrogenic activity of surfactants and some of their degradation products assessed using a recombinant yeast screen. Environ. Toxicol. Chem. 15, 241–248. Routledge, E.J., Sheahan, D., Desbrow, C., Brighty, G.C., Waldock, M., Sumpter, J.P., 1998. Identification of estrogenic chemicals in STW effluent. 2. In vivo responses in trout and roach. Environ. Sci. Technol. 32, 1559–1565. Rudder, J., Van De Wielea, T., Dhooge, W., et al., 2004. Advanced water treatment with manganese oxide for the removal of 17α-ethynylestradiol (EE2). Water Res. 38, 184–192. Streck, G., 2009. Chemical and biological analysis of estrogenic, progestagenic and androgenic steroids in the environment. Trends Anal. Chem. 28, 635–652. Ternes, T.A., Stumph, M., Mueller, J., Haberer, K., Wilken, R.D., Servos, M., 1999. Behavior and occurrence of estrogens in municipal sewage treatment plants – I. Investigations in Germany, Canada and Brazil. Sci. Total Environ. 225, 81–90. Wang, J., Xie, P., Kettrup, A., Schramm, K.-W., 2005. Inhibition of progesterone receptor activity in recombinant yeast by soot from fossil fuel combustion emissions and air particulate materials. Sci. Total Environ. 349, 120–128.
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Analysis of estrogenic activity in environmental waters in Rio de Janeiro state (Brazil) using the yeast estrogen screen Amanda Cristina Vieira Dias a, Frederico Wegenast Gomes a, Daniele Maia Bila b,n, Geraldo Lippel Sant’Anna Jra, Marcia Dezotti a a b
Chemical Engineering Program – COPPE, Federal University of Rio de Janeiro, P.O. Box 68502, 21941-972 Rio de Janeiro, RJ, Brazil Department of Environmental and Sanitary Engineering, State University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil
art ic l e i nf o
a b s t r a c t
Article history: Received 11 November 2014 Received in revised form 7 May 2015 Accepted 10 May 2015 Available online 27 May 2015
The estrogenicity of waters collected from an important hydrological system in Brazil (Paraiba do Sul and Guandu Rivers) was assessed using the yeast estrogen screen (YES) assay. Sampling was performed in rivers and at the outlets of conventional water treatment plants (WTP). The removal of estrogenic activity by ozonation and chlorination after conventional water treatment (clarification and sand filtration) was investigated employing samples of the Guandu River spiked with estrogens and bisphenol A (BPA). The results revealed a preoccupying incidence of estrogenic activity at levels higher than 1 ng L 1 along some points of the rivers. Another matter of concern was the number of samples from WTPs presenting estrogenicity surpassing 1 ng L 1. The oxidation techniques (ozonation and chlorination) were effective for the removal of estrogenic activity and the combination of both techniques led to good results using less amounts of oxidants. & 2015 Elsevier Inc. All rights reserved.
Keywords: Endocrine disrupting chemicals Estrogens YES assay River water quality
1. Introduction Since the 1990s, when the occurrence of endocrine disrupting chemicals (EDCs) influencing the sexual development of fish in English rivers was reported (Purdom et al., 1994), the presence of compounds that potentially interfere with the endocrine system of humans and wildlife has become a major concern worldwide (Streck, 2009). Exposure to EDCs has been associated with damage to the male and female reproductive systems, developmental problems, a decline in the ratio between male and female individuals, changes in the thyroid gland and neurobehavioral effects (Koifman et al., 2002; Pasqualotto et al., 2004; Coleman et al., 2005; James, 2006; Paris et al., 2006). According to Hotchkiss et al. (2008), new concerns have been expressed by scientists regarding the potential role of EDCs in precocious puberty in girls, obesity and type II diabetes in the United States and other countries as well as in relation to complex endocrine alterations induced by mixtures of a variety of potent human and veterinary n Correspondence to: State University of Rio de Janeiro (UERJ), Department of Sanitary and Environmental Engineering, Rua Sao Francisco Xavier 524, 5029-F, CEP 20550-900 Rio de Janeiro, RJ, Brazil. E-mail addresses: [email protected] (A.C.V. Dias), [email protected] (F.W. Gomes), [email protected] (D.M. Bila), [email protected] (G.L. Sant’Anna Jr), [email protected] (M. Dezotti).
http://dx.doi.org/10.1016/j.ecoenv.2015.05.013 0147-6513/& 2015 Elsevier Inc. All rights reserved.
pharmaceutical products, personal care products, nutraceuticals and phytosterols present in the environment. Endocrine disruptors, such as estrogenic chemicals, are introduced into the aquatic environment by human activity, both domestic and industrial. In developed countries, effluents from municipal wastewater treatment plants (WWTPs) are the main source of estrogenic chemicals in natural waters, because these substances are not completely removed in conventional treatment processes (D’Ascenzo et al., 2003; Auriol et al., 2006). In developing countries, like Brazil, the problem seems to be more acute, since wastewaters without previous treatment are commonly released into the environment and this is certainly the major source of estrogenic compounds in surface waters. The Brazilian population (203 million inhabitants) is concentrated in urban areas. The resulting intensive soil occupation coupled with inadequate infrastructure for sewage collection and treatment has a negative effect on the quality of natural waters. Despite the fact that Brazil has abundant sources of fresh water and high-capacity water treatment plants (WTPs), such as that located in the metropolitan area of Rio de Janeiro (the largest WTP in the world, with the capacity to treat continuously 43 m3/s of raw water), few studies on the occurrence of estrogenic compounds in local natural water sources could be found in the literature (Ternes et al., 1999; Araújo 2005; Ghiselli, 2006; Raimundo CCM, 2007; Gerolin ERR, 2008; Reis Filho, 2008;
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Kuster et al., 2009). Among the cited authors, only Kuster et al. (2009) evaluated water samples collected from the main water supply sources of Rio de Janeiro State. Reis Filho (2008) discussed the biological effects associated with the presence of estrogenic compounds in natural waters in Sao Paulo State. The presence of estrogenic compounds in river samples was observed in both studies: in the former the occurrence of phytoestrogens, both free and conjugated estriol (E3), and conjugated 17β-estradiol (E2) in natural samples was reported and in the latter strong evidence of endocrine disruption in male fish due to the presence of estrogenic compounds and the presence of 17α-ethinylestradiol (EE2) in some river samples were verified. It is widely known that in chemical analysis the interactions in mixtures are not taken into consideration and, consequently, accessing the biological effects of the water matrix will require the use of other types of assays (Routledge and Sumpter, 1996). The yeast estrogen screen (YES) is one of the most common in vitro bioassays used to provide initial evidence of estrogenic contaminants in ecosystems. This is a robust, rapid and sensitive tool for assessing estrogenic activity in environmental samples at moderate cost (Beck et al., 2006). The YES assay is used to evaluated the estrogenic activity in waters and wastewaters (Brix et al., 2010; Li et al., 2014). The lack of information regarding endocrine disruption activity caused by the presence of estrogenic compounds in Brazilian natural waters and treated water for public supply motivated us to carry out this study. The YES assay was used to assess the estrogenic activity in natural waters in Rio de Janeiro State (Paraíba do Sul-Guandu River system). The location of the aquatic environment investigated is shown in Supplementary information, Fig. S1 Samples were collected in six sampling campaigns performed between March 2007 and December 2008. The determination of the estrogenicity of both raw and treated water samples was also included in the experimental work. Moreover, the estrogenic activity in water samples collected from the Guandu River was monitored and its removal during oxidative treatment by ozonation and chlorination was evaluated in bench-scale experiments performed in our laboratory. The aim of this research was to contribute to the determination of the degree of pollution in important aquatic environments in Rio de Janeiro State and evaluate the potential of ozonation and chlorination to reduce the estrogenic activity.
2. Materials and methods 2.1. Reagents Stock solutions of 17β-estradiol (E2) and 17α-ethinylestradiol
(EE2) (98% purity, Sigma-Aldrich) were prepared at 100 mg L 1 in acetone and stored at 4 °C. All medium constituents were obtained from Sigma-Aldrich except biotin, which was supplied by Merck. Purified water was obtained from a Milli-Q Biocell system (Millipore). Chlorophenol red-β-D-galactopyranoside (CPRG) was supplied by Roche Diagnostics GmbH. For the sample extraction, hexane, methanol and acetone (HPLC and spectrophotometric grade solvents) were supplied by Tedia Brazil and ethanol (HPLC grade) and BPA were obtained from Merck. 2.2. Area of study The state of Rio de Janeiro, whose capital (the city of Rio de Janeiro) was chosen to host the 2016 Olympics Games, is located in southeastern Brazil and has around 15.2 million inhabitants (IBGE, 2010a). Approximately 6.3 million live in the central city area of Rio de Janeiro and 12.4 million in the wider metropolitan area. Other important towns in this state, which lie far from the capital, are Campos dos Goytacazes, Resende and Volta Redonda. The Guandu and Paraiba do Sul Rivers are the most important sources of water in the state of Rio de Janeiro, since together they supply more than 12 million people, including 85% of the metropolitan area residents. The Paraiba do Sul & Guandu system supplies electric power and water for several cities. A flow rate of 160 m3 s 1 is diverted from the Paraiba do Sul River to the Guangu River to assure water supply for the city of Rio de Janeiro. All sampling locations are shown in Supplementary information, Fig. S1, highlighted by black circles which in the case of sampling locations 3 and 5 represent more than one sampling site. Sampling dates, site codes and the respective GPS coordinates are listed in Table 1. The sampling sites 1, 2 and 3 are located in medium-sized cities and represent the most populated and industrialized region along the Paraiba do Sul River in the studied area. Site 1 is the closest to the boundary between the states of Sao Paulo and Rio de Janeiro and represents the quality of the Paraiba do Sul River as it flows into Rio de Janeiro State. At site 3, samples of water from the Paraiba do Sul River treated in a WTP (Tw3 samples) were also collected during all sampling campaigns carried out in 2008. Sampling site 4 is located in the north of the state, along the estuary of the Paraiba do Sul River, in the town of Campos dos Goytacazes. In this region agriculture and agro-industry are important economic activities. Sampling site 5 is located in Nova Iguacu where water from the Guandu River is driven to the water treatment plant and subsequently supplied to Rio de Janeiro metropolitan area. At this location, treated and raw water samples were collected. The water
Table 1 Description of water sampling sites in selected areas of Rio de Janeiro State. CodeFig. 1
Location (inhab.)a
1 2 3
Resende (130,035) Barra Mansa (176,899) Volta Redonda (261,404)
4 5
C. Goytacazesb (431,839) Nova Iguaçu
a b c
Samplecode
Source
PS1 PS2 PS3 Tw3c PS4 G1 G2
Paraiba do Sul Paraiba do Sul Paraiba do Sul Tap water Paraiba do Sul Guandu river Guandu river
Tw5c
Tap water
GPS coordinates
river river river river
o
o
22 .27.94’S44 .26.90’W 22o.31.50’S 44o.11.33’W 22o.30.05’S44o.05.42’W 22o.30.11’S44o.05.44’W 21o.44.47’S 41o.20.16’W 22o.49.28’S43o.37.31’W 22o.48.70’S43o.37.71’W 22o.49.76’S43o.36.99’W
Sampling data and campaigns 2007
2008
Mar. Apr. Mar. Apr. Mar. Apr. Oct.
Jan. Jan. Jan. Jan.
Mar. Apr Mar. Apr. Sep.
Jul. Jul. Jul. Jul.
Sep. Sep. Sep. Sep.
Jan. Jun. Jul. AugSep. Oct. Nov. Dec. Mar. Apr. Sep. Jan. Aug. Nov
IBGE (2009). Campos dos Goytacazes. Treated water from different WTP (primary treatment processes: coagulation and flocculation, sand filtration and chlorination).
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samples from the Guandu River were initially collected along its left margin (G1 samples). However, the sampling location was transferred to a nearby bridge, close to the Guandu WTP inlet (G2 samples), in order to evaluate the estrogenic activity at the point of greatest clearance (far from the points of contamination along the river margins). Samples of the water treated at the Guandu Water Treatment Plant were also collected at the exit of the plant (Tw5) and the estrogenic activity was determined. 2.3. Estrogenicity removal by ozonation and chlorination All samples were filtered and concentrated by solid phase extraction before evaluation of the estrogenic activity and data were expressed as 17β-estradiol (E2) equivalents (E-EQ). To simulate the water treatment performed in local WTPs, coagulation was conducted using FeCl3 followed by sedimentation in a jar-test apparatus as follows: ferric chloride dose of 35 mg L 1, strong agitation for 5 min, mild agitation for 20 min and sedimentation for 30 min. The supernatant was then filtered through a sand filter (bed height of 25 cm) containing particles of sizes ranging from 0.4 to 0.9 mm. The water sample was then filtered through 1.2 μm glass fiber filters and chlorinated (NaClO) and/or ozonated. Ozonation was carried out in a system comprised of a glass contact column (1 L), an ozone generator (Unitek – model UTK) and an ozone analyzer (IN USA, ASX-Mod H1) for the gas phase. Ozone doses in the range of 0.5–3.5 mg O3 L 1 were applied. Chlorination was conducted using chlorine doses of 1.5 and 3.2 mg Cl2 L 1 All experiments were performed at 247 2 °C and pH was in the range of 6–7. Since the estrogenic activity in the Guandu River water was low, to investigate the potential of lab-scale chlorination and ozonation to remove estrogenic activity, samples were spiked with E2, EE2 and BFA to give the following concentrations: E2 (2.5 and 5 ng L 1), EE2 (2.5 and 5 ng L 1) and BFA (0, 500 and 1000 ng L 1). 2.4. Water quality: analytical determinations All aqueous samples (5 L) were collected in acetone-rinsed amber glass bottles and returned to the laboratory where they were immediately characterized by measuring the dissolved organic carbon (DOC), turbidity, pH, conductivity and N-NH3, according to APHA (2005). 2.5. Sample preparation for the estrogenic activity assay All samples were filtered through 1.2 μm glass fiber filters (Millipore) immediately after collection. Aliquots (1 L) of the samples collected in 2007 and in January 2008 were also filtered through 0.45 μm cellulose acetate filters (Sartorius, Göttingen, Germany) and 0.22 μm glass fiber filters (MFS, USA) in parallel, and the estrogenic activity of each filtrate was determined. The filtered samples were acidified to pH 3 and stored at 4 °C until extraction, which was performed within 48 h after sampling. After the filtration, all samples were concentrated by solid phase extraction (SPE) before the YES assay. SPE was performed using 500 mg and 2 g Bond Elut C18 cartridges (Varian) for samples filtered through both 0.22 μm and 0.45 μm filters and for samples filtered through a 1.2 μm filter, respectively. Immediately before use the cartridges were conditioned with hexane, acetone and methanol and washed with purified water at pH 3. The prefiltered samples were forced under vacuum through the cartridge at a flow-rate of approximately 10 mL min 1. The cartridges were kept under vacuum aspiration for 30 min after the extraction had finished. The elution of analytes from the cartridges was performed using 4 mL of acetone.
43
The solvent was evaporated under a gentle nitrogen stream to dryness. Finally, the dry samples were reconstituted with 2 mL of ethanol and stored at 18 °C until analysis applying the YES assay. 2.6. Yeast estrogen assay The estrogenic activity of the sample extracts was determined using a recombinant reporter gene assay known as yeast estrogen screen (YES). This recombinant Saccharomyces cerevisiae cell line contains the human estrogen receptor gene linked to a receptor gene coding for β-galactosidase and was kindly provided by Prof. Sumpter, Brunel University, UK. In the presence of estrogenic chemicals, this enzyme is produced and secreted into the assay medium, where it breaks down the chromogenic substrate CPRG, and the color change is quantified. Yeast cells were cultured and the assay was carried out as described by Routledge and Sumpter (1996) with some modifications. The E2 standard solution and sample extracts were serially diluted in ethanol and 10 μL of each dilution were transferred (at least in duplicate) into a 96-well optically flat microtiter plate and allowed to evaporate until dryness. Next, 200 μL of seeded yeast medium containing CPRG were added to the wells. The plates were then sealed with autoclave tape and shaken vigorously for 2 min. After incubation for 72 h at 30 °C the absorbance was read at 540 nm (for color) and at 620 nm (for turbidity) using a plate reader (BIO-TEK EL808). In each test, ethanol and E2 (serially diluted) were used as blank solvent control and positive control, respectively. 2.7. Data analysis The estrogenic responses for the sample extract and E2 standard were plotted after being corrected for turbidity (Coleman et al., 2004). Inhibition of yeast cell growth was taken as an acute toxic effect of a tested sample and was observed a reduction in the absorbance at 620 nm, compared to the reference wells (i.e., solvent control) (Aerni et al., 2004; Beck et al., 2006). Calibration dose–response curves for E2 standard were plotted giving concentration versus estrogenic response in corrected absorbance at 540 nm. The resulting sigmoidal curves were fitted to a symmetric logistic function (Origin 6.0, Microsoft, USA). The estrogenic activity in each well containing sample extract was then calculated as E2 equivalents (E-EQ) by interpolation from the E2 standard curves (in μg L 1). These values were divided by the SPE concentration factor, resulting in the final concentration for the water sample as E-EQ (range of ng L 1). In this study, dose–response curves for E2 were obtained for a concentration range of 1.36 μg L 1 to 0.67 ng L 1 in the wells (Rudder et al., 2004; Fine et al., 2006).
3. Results and discussion 3.1. Water quality The characteristics of the water samples collected in different places are shown in Table 2. The dissolved organic carbon (DOC) was between 3 and 16.6 mg L 1 and for most samples it was lower than 7 mg L 1. Turbidity ranged from 3.4 to 25.2 NTU and the pH was close to 7 for all samples. Levels of N-NH3 were, in general, low (between 0.2 and 1.1 mg L 1) and water conductivity was below 100 mS cm 1 with the exception of one sample for which the conductivity surpassed this upper limit.
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Table 2 Water quality of samples collected in different river locations. Source
Location
Sample code
Sampling date
DOC (mg L 1)
Turbidity (NTU)
pH
Conductivity (mS cm 1)
N-NH3 (mg L 1)
PS Rivera
Resende
PS1
Barra Mansa
PS2
Volta Redonda
PS3
Campos Goytacazes
PS4
3/15/2007 4/16/2007 1/21/2008 7/23/2008 9/30/2008 3/15/2007 4/16/2007 1/21/2008 7/23/2008 9/30/2008 3/15/2007 4/16/2007 10/2/2007 1/21/2008 7/23/2008 9/30/2008 3/20/2007 4/18/2007
8.5 4.6 3.7 8.5 6.4 5.4 3.0 – 6.7 4.2 10.4 4.4 6.0
10.2 8.2 19.4 3.4 5.3 11.0 10.2 25.2 4.4 6.0 12.7 8.0 5.9 24.0 4.4 7.5 21.7 25.4
6.7 6.8 6.5 7.2 7.2 6.5 7.4 6.6 7.4 7.3 6.7 7.0 6.7 6.6 7.2 7.3 7.0 7.0
79.0 85.3 91.9 86.7 86.1 76.5 78.5 72.3 84.5 86.3 80.6 103.3 98.6 86.4 83.9 94.5 61.1 64.4
0.2 0.2 0.6 0.6 0.4 0.2 0.5 0.4 0.6 0.4 0.3 0.3 1.5 0.7 1.1 0.6 0.4 0.2
Guandu River
Nova Iguaçu
G1
3/15/2007 4/16/2007 9/27/2007 1/28/2008
6.4 16.6 5.9
16.3 8.1 4.5 17.3
6.8 6.7 6.9 6.6
77.6 84.6 96.2 86.1
0.3 0.2 2.6 0.4
Guandu River
Nova Iguaçu
G2
7/1/2008 7/9/2008 8/1/2008 8/19/2008 8/27/2008 9/17/2008 10/9/2008 10/22/2008
6.7 5.6 3.9 4.3 4.6 3.1 4.5 3.0
5.4 5.3 4.5 4.6 5.0 4.7 24.2 8.2
6.8 7.0 7.0 7.5 7.6 7.2 7.1 7.4
– – 88.7 94.8 96.9 96.2 85.6 81.8
0.4 0.5 03 – 0.8 0.4 – 1,1
a
Paraiba do Sul River.
3.2. Estrogens in Rio de Janeiro state water sources The sanitation conditions in many municipalities in the southeast region of Brazil can be described as poor. Data for 2008 reveal that, out of 1668 municipalities in this region, 1586 have sewage collection systems in operation. However, only 808 municipalities have sewage treatment facilities and treatment of sewage at a secondary level (biological) is only performed in 461 municipalities (IBGE, 2010b). Thus, in many localities sewage reaches the aquatic systems without any type of treatment or is precariously treated. The estrogenic activity results are shown in Fig. 1. The seasonal variations will have an effect on the results, particularly the E-EQ (ng/L) 15
10
03/15/07 04/16/07 01/21/08 07/23/08 09/30/08
03/15/07 04/16/07 01/21/08 07/23/08 09/30/08
03/15/07 04/16/07 10/02/07 01/21/08 07/23/08 09/30/08
03/20/07 04/18/07
03/15/07 04/16/07 09/27/07 01/28/08
07/01/08 07/09/08 08/01/08 08/19/08 08/27/08 09/17/08 10/09/08 10/22/08
5
PS1
PS2
PS3
PS4
G1
G2
Fig. 1. Estrogenic activity results: white bars relate to samples filtered through 0.45 μm membrane and black bars to samples filtered through 1.2 μm membrane.
raining periods in summer (December to March). In Fig. 1, white and black bars correspond to samples filtered through 0.45 and 1.2 μm filters, respectively. Since samples filtered through 0.22 μm and 0.45 μm membranes led to almost the same results, only data from the latter case are reported herein. Since adverse effects of estrogens have been observed at 1 ng L 1 (Routledge et al. 1998, Petrovic et al., 2002), this value was taken as a reference in this study and is represented by the dashed line in Figs. 1 and 2. The limit level of 1 ng L 1 was also considered by Matthiessen. et al. (2006) in their work with livestock farms streams. The data shown in Fig. 2 reveals that values far higher than this limit were observed in some samples from all sampling points. Several samples filtered through a 1.2 μm filter showed notably high E-EQ values. In order to obtain a more consistent view of the E-EQ results, values surpassing 1 ng L 1 and their incidence are reported in Table 3 for all sampling campaigns. Values of E-EQ lower than 1 ng L 1 were observed for samples Resende (PS1), Volta Redonda (Tw3) and C. Goytacazes (PS4) filtered through a 0.45 μm filter and also for Volta Redonda (Tw3) for samples filtered through a 1.2 μm filter. In general, the estrogenicity of the river water samples seems to increase as the Paraiba do Sul River flows through the Rio de Janeiro State and reaches maximum values at Volta Redonda (PS3). For samples collected from the Guandu River (G1 and G2) 8% of the E-EQ values were higher than 1 ng L 1. However, a matter of concern is the high incidence (25%) of water samples treated by conventional techniques (Tw5, filtered through 1.2 μm) presenting E-EQ values which surpass this limit. The high incidence (50%) observed for samples collected from C. Goytacazes (PS4) and filtered through a 1.2 μm filter should be considered with prudence, since the number of samples collected at this site was small. The presence of estrogens and progestogens in the Paraiba do
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contrast to estradiol 17-glucoronide and estriol 16-glucoronide which do not give any response in the assay. However, these glucoronides are easily converted to estriol and estradiol by enzymes produced by bacteria found in sewage and waters. Thus, these substances and the non-conjugated hormones (estriol and estradiol), which were also detected by Kuster et al. (2009), could be responsible for the estrogenic activity found in detectable and significant levels in the aquatic system investigated in the study reported herein, which receives important amounts of non-treated sewage.
E-EQ (ng.L-1) 15
10
10.3
9.7
9.0
5
0.9
RWs
O (3.5 mg.L-1 )
CFW
CW
45
3.3. Estrogenicity removal by chlorination and ozonation E-EQ (ng.L-1) 15
10
5
5.6
4.8
3.2 0.8
RWs
CW
O3 (1 mg.L-1)
CFW
0.9
0.3
O3 (3.5 mg.L-1)
O3 (2 mg.L-1)
E-EQ (ng.L-1) 15
10
5 2.3
2.7 1.3
RWs CW
0.3
0.3
O3 O3 O3 (0.5 mg.L-1) (1 mg.L-1) (2 mg.L-1)
0.4
0.2
Cl2 Cl2 (1.5 mg.L-1) (3.2 mg.L-1)
0.1
Cl2 (1.5 mg.L-1) O (0.5 mg.L-1)
Fig. 2. Estrogenic activity of Guandu River water spiked with: (a) E2, EE2 (5 ng L 1) and BPA (1000 ng L 1); (b) and (c) E2, EE2 (2.5 ng L 1) and BPA (500 ng L 1). All samples were treated by clarification, sand filtration and ozonation. RWs – River water (spiked), CW – Clarified water, CFW – Clarified and filtrated water.
Sul and Guandu Rivers has been previously detected by Kuster et al. (2009). These authors found appreciable levels of progesterone in samples collected from Volta Redonda, Barra Mansa and Guandu River. In addition, estriol, estradiol 17-glucoronide and estriol 16-glucoronide were detected in water samples collected from these locations. In the treated Guandu River water, estradiol 17-glucoronide and progesterone were also detected. Progesterone, possibly due to its chemical structure, gives an estrogenicity response in the YES assay (Wang et al., 2005) in
In previous studies carried out in our laboratory ozone was able to remove the estrogenicity associated with 17β-estradiol (Bila et al., 2007) and that related to both 17β-estradiol and 17α-ethinylestradiol (Maniero et al., 2008) in pure water. The results obtained motivated us to investigate ozonation alone or in combination with chlorination in order to remove or reduce estrogenic EDCs from a more complex matrix (river water). The Guandu River water was treated in the laboratory, as described in Section 2.3. The treatment was able to reduce turbidity to a large extent, as shown by the data in Table 4. The pH of the water samples dropped slightly after treatment and remained in the range of 6.0–6.6. Table 4 summarizes the experimental conditions used in ozonation and chlorination experiments. In the last three experiments ozonation and chlorination were applied in the water treatment. Fig. 2a shows the results obtained in assays conducted with the highest concentrations of E2 and EE2 (5 ng L 1) and BFA (1000 ng L 1). The estrogenicity of the water spiked with these substances was not removed after clarification and sand filtration. However, it was reduced to an average value of 0.9 ng L 1 E-EQ after ozonation (3.5 mgO3 L 1). Fig. 2b shows similar results for initial E2, EE2 and BFA concentrations of 2.5, 2.5 and 500 ng L 1, respectively. A pronounced estrogenicity reduction was enhanced by ozonation at the three ozone doses tested. In experiments without BFA and with E2 and EE2 concentrations of 2.5 ng L 1, ozonation and chlorination alone or combined were investigated. As shown in Fig. 2c, ozone doses of 1 and 2 mgO3 L 1 were able to reduce estrogenic activity to an average value of 0.3 ng L 1 E-EQ. Chlorination was also effective in the removal of estrogenicity using doses of 1.5 and 3.2 mgCl2 L 1. However, an interesting result was obtained combining a low ozone dose (0.5 mgO3 L 1) with a low chlorine dose (1.5 mgCl2 L 1). This condition led to the lowest residual estrogenic level in the water (0.1 ng L 1). Thus, the combination of chlorination and ozonation seems an interesting alternative to remove estrogenicity and reduce the use of oxidants. The results obtained in this study revealed the presence of
Table 3 Maximum E-EQ values and incidence of values higher than 1 ng/L. Location
Sample code Sampling date
Maximum E-EQ (ng L 1) f o 0.45 μm
Resende Barra Mansa
PS1 PS2
0.9 1.7
Volta Redonda Volta Redonda Nova Iguaçu Nova Iguaçu Nova Iguaçu C.Goytacazes
PS3 Tw3 G1 G2 Tw5 PS4
3.1 ndl 1.3 0.5 0.9 0.5
03/15/2007 09/30/200803/15/ 2007 03/15/2007 01/21/2008 03/15/2007 10/22/2008 03/15/2007 03/20/2007
Maximum E-EQ (ng L 1) f o1.2 μm
E-EQ41 ng/L incidencen (%)
0 9
2.9 4.8
20 20
17 0 25 0 0 0
16 ndl 15 2.3 10 1.3
50 0 8 13 25 50
E-EQ41 ng/L incidencen (%)
ndl ¼ non detectable levels. n
ncidence ¼ (number of samples with E-EQ 4 1 ng/L).100/(total number of samples).
46
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Table 4 Experiments with Guandu River water spiked with different concentrations of E2, EE2 and Bisphenol A (BFA). River water and treated water characteristics
Oxidation experiments conditions
Sampling Date
River water pH turbidity (NTU)
Treated water pH turbidity (NTU)
Ozone and/or chlorine doses and initial concentrations
09/12/2008 09/19/2008 10/22/2008 11/14/2008 12/11/2008 12/17/2008
7.0 7.2 7.4 7.5 7.4 7.5
6.2 6.0 6.2 6.3 6.6 6.4
3.5 mgO3 L 1; E2 and EE2 (5 ng L 1), BFA (1000 ng L 1) 3.5 mgO3 L 1; E2 and EE2 (2.5 ng L 1), BFA (500 ng L 1) 1 and 2 mgO3 L 1; E2 and EE2 (2.5 ng L 1), BFA (500 ng L 1) 0.5; 1 and 2 mgO3 L-1; 1.5 and 3.2 mgCl2 L-1; E2 and EE2 (2.5 ng L-1) 0.5; 1 and 2 mgO3 L 1; 1.5 and 3.2 mgCl2 L 1; E2 and EE2 (2.5 ng L 1) 0.5 and 1 mgO3 L 1; 1.5 mgCl2 L 1; E2 and EE2 (2.5 ng L 1)
4 4 11 16 18 34
0.02 0.02 0.10 0.14 2.1 2.2
estrogenic activity in the river water samples and also in samples collected downstream of the water treatment plants (WTPs). Although chlorination is a necessary and effective process in WTPs, mainly in tropical countries due to the high temperatures and the extension of the water distribution system, it should be combined with other processes to assure estrogenicity removal. The results reported herein highlight the need to improve the technology used in WTPs, chiefly in countries which have emerging and important economies, like Brazil.
agencies (CNPq and FAPERJ).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at
References 4. Conclusions The estrogenic activity of river waters (Paraiba do Sul and Gundu Rivers) is a matter of concern. For samples filtered through 1.2 μm filters, the estrogenic activity increased as the Paraiba do Sul River flowed through the Rio de Janeiro State. Maximum values of 2.9, 4.8 and 16 ng L 1 E-EQ were observed in samples collected at Resende, Barra Mansa and Volta Redonda, respectively. The percentage of samples (20–50%) collected from these sites which presented estrogenic activity was also a matter of concern. The discharge of raw and treated sewage into the river is common in cities due to deficient sanitation and treatment systems. In the case of the Guandu River, the incidence of samples presenting estrogenic activity higher than 1 ng L 1 E-EQ was 8%. However, very high maximum values (15 ng L 1 E-EQ) were observed for samples filtered through a 1.2 μm filter and a preoccupying result was the incidence of 25% of samples presenting activity higher than 1 ng L 1 after the treatment of Guandu River water by conventional techniques. The results obtained in this study demonstrate that water contamination by endocrine disrupting chemicals is occurring at several locations along the Paraiba do Sul and Guandu Rivers. Furthermore, estrogenic activity was observed in water supply systems, indicating that water treatment plants should be redesigned in order to achieve the removal of endocrine disrupting chemicals. The results for the Guandu River water treated by chemical clarification, sand filtration and ozonation and/or chlorination were promising, since ozonation was effective in reducing the estrogenicity levels of 5 and 2.5 ng L 1 E-EQ to values far below the reference value of 1 ng L 1. In addition, the combination of low ozone and chlorine doses (0.5 mgO3 L 1 and 1.5 mgCl2 L 1) promoted a considerable reduction in the estrogenic activity using relatively low doses of oxidants. In developing countries the chlorination process is already applied in WTPs but ozonation is not widely used. Thus, investments in ozonation or other processes that are effective for the removal of EDCs are required to ensure the supply of water free from these micropollutants.
Acknowledgements Authors express their gratitude to the following Brazilian
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