Estrogen pollution in a highly productive ecosystem off central-south Chile

Marine Pollution Bulletin 62 (2011) 1530–1537

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Estrogen pollution in a highly productive ecosystem off central-south Chile Angéline Bertin a,b,⇑, Pedro A. Inostroza a, Renato A. Quiñones a,c a

Programa de Investigación Marina de Excelencia (PIMEX-Nueva Aldea), Universidad de Concepción, Casilla 160-C, Barrio Universitario s/n, Concepción, Chile Departamento de Biología, Facultad de Ciencias, Universidad de La Serena, Casilla 599, La Serena, Chile c Centro de Investigación Oceanográfica en el Pacífico Sur Oriental (COPAS), Universidad de Concepción, Casilla 160-C, Barrio Universitario s/n, Concepción, Chile b

a r t i c l e

i n f o

a b s t r a c t

Keywords: Steroid estrogen Coast Sediment Central-south Chile

While the presence of steroid estrogens in the environment has become a major environmental and health concern, their occurrence in coastal sediments remains poorly characterized. In this study, we measured the levels of three natural (estrone, 17b-estradiol, estriol) and one synthetic (17aethinylestradiol) estrogens in 54 coastal sediment samples collected from nine locations off central-southern Chile. Steroid estrogens were found in every sample. Remarkably high levels of 17a-ethinylestradiol were detected, reaching up to 48.14 ng/g dry weight. As a result, the global estrogenic loads were estimated to be high at all sites. Interestingly, they were found to correlate with the size of human populations served by sewage plants. Our study indicates that 17a-ethinylestradiol may accumulate in coastal sediments. The possible impact of this highly potent synthetic estrogen on the biota of the marine ecosystem off central-south Chile and on human health remains an open question. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction

and considerable attention has been paid to examining the potential repercussions of these compounds in sewage impacted areas (Carballo et al., 2005; Jobling et al., 1998, 2002a,b). Monitoring studies have demonstrated that contamination by steroid estrogens in aquatic environments receiving STW effluents is widespread (Desbrow et al., 1998; Johnson and Sumpter, 2001). The adverse effects of this type of pollution on local wildlife are substantiated by laboratory studies indicating that steroid estrogen levels in such areas are usually high enough to disrupt normal functioning of aquatic animal endocrine systems (Desbrow et al., 1998; Thorpe et al., 2000; Cargouet et al., 2007), as well as by field studies demonstrating that endocrine disruption in wild fish correlates with predicted levels of exposure to steroid estrogens derived from human populations (Jobling et al., 2006). Although mounting evidence indicates that steroid estrogens threaten all kinds of aquatic ecosystems – inland systems, (Baronti et al., 2000; Cargouet et al., 2007; Aerni et al., 2004; Pojana et al., 2004; Chen et al., 2007; Kolok et al., 2007; Kuch and Ballschmiter, 2001; Lagana et al., 2004; Petrovic et al., 2002b; Vigano et al., 2006, 2008; Yamamoto et al., 2006; Lopéz de Alda et al., 2002) and marine systems (Atkinson et al., 2003; Beck et al., 2005; Braga et al., 2005b; Saravanabhavan et al., 2009; Isobe et al., 2006) – relatively little is known about hormonal pollution in coastal sediments (but see Braga et al., 2005b; Isobe et al., 2006; Zhang et al., 2009). Yet, sediment contamination by steroid estrogens may be of paramount importance in such habitats. Indeed, the high ionic strength of seawater may enhance sorption of certain steroid estrogens onto sediment particles (Robinson et al., 2009; Bowman et al., 2002); and, particularly of the most hydrophobic ones, like the highly potent

In recent decades, there has been increasing evidence that chemicals with the ability to disrupt animal endocrine systems have contaminated aquatic environments (WHO, 2002). Because these compounds, named endocrine disrupting compounds, can cause dramatic developmental and/or reproductive dysfunctions (WHO, 2002), they have become a worldwide concern and are regarded as one of the most serious anthropogenic threats to biodiversity and ecosystems (Jenssen, 2006), and to human health (Diamanti-Kandarakis et al., 2009). Steroid estrogens are, in this context, of great concern since they have emerged as the most probable common cause of endocrine disruption observed in wild fish (Johnson and Williams, 2004). Indeed, these compounds are by far the most potent endocrine disrupting compounds recognized (for review see Caliman and Gavrilescu, 2009), and because they are excreted by humans and animals, they are likely to threaten aquatic biota and public health in many parts of the world (WHO, 2002). The presence of natural and synthetic estrogens in effluents discharged by human sewage treatment works (STWs) is well established (Ahel et al., 1994; Purdom et al., 1994; Jobling et al., 1998; Baronti et al., 2000; Petrovic et al., 2002b; Jobling and Tyler, 2003; Sumpter, 2005; Braga et al., 2005c; Robinson et al., 2009), ⇑ Corresponding author at: Departamento de Biología, Facultad de Ciencias, Universidad de La Serena, Casilla 599, La Serena, Chile. Tel.: +56 51 334638; fax: +56 51 204383. E-mail address: [email protected] (A. Bertin). 0025-326X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2011.04.002

A. Bertin et al. / Marine Pollution Bulletin 62 (2011) 1530–1537

synthetic compound, ethinylestradiol (Robinson et al., 2009). This kind of phenomenon could explain why sediment estrogenic activities are increasingly reported in marine and coastal areas (Hashimoto et al., 2005; Houtman et al., 2007; Legler et al., 2003; Oh et al., 2000; Qiao et al., 2009; Schipper et al., 2009). Field data from marine systems are necessary to attest to this. They are also essential in order to both predict the persistence of steroid estrogens in a given environment (Lartiges and Garrigues, 1995) and to determine the risks consequently imposed on local wildlife (Lai et al., 2002). In this study, we evaluate the burden that steroid estrogens might cause upon a major fishing zone off central-southern Chile by measuring, in sediments impacted by human sewage, the concentrations of three natural estrogens: estrone (E1), 17b-estradiol (E2), estriol (E3), and of 17a-ethinylestradiol (EE2), a major constituent of female contraceptives. The coastal region of interest houses a large variety of fish and seafood of commercial value such as flatfish, hake, red and pink cusk-eels, drumfish, crabs, mussels, abalone, and clams (Sernapesca, 2008). Thus, understanding whether steroid estrogen contamination in Chilean coastal sediments threatens the health and/or existence of local aquatic biota will not only improve our understanding of the real ecological and

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sanitary threat posed by discharges from human sewage in coastal environments, but will also be of interest from ecological, commercial and human health perspectives. 2. Materials and methods 2.1. Sampling location and procedure Sediment samples were collected in early 2009 at the discharge locations of nine STWs distributed along the Pacific Ocean coastline of the Biobío Region (central-southern Chile, Fig. 1). The geographical coordinates of the discharge locations were obtained by consulting declarations of STW environmental impact assessments published online by the Sistema de Evaluación de Impacto Ambiental of Chile (SEIA) and a Global Positioning System device was used to locate and collect sediments near the sewage outfalls. The principal characteristics of the STWs are presented in Table 1. All of them receive wastewaters of domestic origin and four additionally carry out the treatment of industrial wastewaters. Most of the STWs considered here provide primary treatment only and their discharges are released directly into the Pacific Ocean (Table 1).

Fig. 1. Study zone and sampling sites.

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Table 1 Principal characteristics of the STWs discharging their treated effluents in the sampling area. City

Size of the served population

Average flow (mm3/ day)

Biological treatment

Wastewater origin

Dichato Tomé Penco Talcahuano

13,129 45,959 45,361 144,619

2.3a/0.6 4.1 5.2 31.0

Yes No No No

44,813

84.1

No

392,827 80,447 92,384

86.4 17.4 4.7

Yes No No

3.6

Yes

Domestic Domestic Domestic Domestic + industrial (fish meal industries) Domestic + industrial (fish meal, oil production, iron and steel, and chemical industries) Domestic Domestic Domestic + Industrial (fish meal industries) Domestic + industrial (paper and pulp mills)

San Vicente

Concepción San Pedro Coronel Arauco a

24,269

From December to February only.

Six sediment samples were collected at each site. They were obtained from two separate field trips performed in January and March 2009. At each sampling station, sediment samples (150 g) from the top 10 cm of sediment were collected in triplicate at all sites, using either a Van Veen drag or a sediment core sampler. Each sample was wrapped in aluminum foil, kept on ice in a cooler during transport and then stored at 20 °C until extraction. 2.2. Sediment sample preparation and gas chromatography–mass spectrometry (GC–MS) analysis Sediment sample preparation was based on the procedure described in Peng et al. (2006). Analytes were extracted from the sediments via ultrasonication. Twenty grams of wet sediment (mean ± SD water content: 37.59% ± 14.44) were dissolved into a 20 mL mixture of acetone/dichloromethane (1:1) and then sonicated during 15 min (Bransonic Ultrasonic Cleaner 5510, 50– 60 Hz, 220 V, 2A). Sonication was repeated four times and the extracts were combined into a glass separatory funnel containing 30 mL of dichloromethane and 10 mL of sodium chloride solution. After vigorous shaking of the aliquots, the organic phase was recovered and dried out by rotary evaporation. Prior to GC–MS determination, the extracts, redissolved in 1 ml of hexane, were fractionated using silica gel columns with a hexane–dichloromethane–methanol gradient. After being dried-out by rotary evaporation and then under a gentle flow of nitrogen, the methanol eluent was derivatized at 60 °C for 30 min with 100 lL N,O-bis(trimethylsilyl) trifluoro acetamide and 5% trimethylchlorosilane/pyridine (1:1). The eluent was once again dried out before being redissolved in hexane (1 ml). Steroid estrogen measurements were made by injecting 1 lL of this extract into an Agilent GC 7890A system connected to an Agilent 5975C mass detector operating in electron impact mode at 70 eV. The gas chromatograph was equipped with a HP-5MS (30 m  0.25 mm  0.25 lm) capillary column using He at a flow rate of 1.5 ml/min as a carrier gas. The GC oven temperature was programmed at 60 °C for 1 min and then ramped up 10 °C/min to 290 °C, and held at this temperature for 15 min. 2.3. Quality assurance and quality control Column performance was checked for each steroid estrogen analysed by running standards, acquired from Sigma–Aldrich

(Oakville, ON, USA). Before each set of samples, 1 lL of hexane was injected to discard potential contamination of the column. Procedural blanks using diatomaceous earth (Dionex ASEÒ Prep, DE) and purified sea sand (Merck) were analyzed alongside standards and samples. None of the target compounds were detected in the blanks. Identification of the analytes was achieved by selected ion monitoring of m/z 192–218–257–285–342 for E1, 129– 205–232–285–416 for E2, 311–345–386–414–504 for E3, and 196–232–285–425–442 for EE2. For each steroid, standard curves were prepared using estrogen standards. The quantification was realized using a calibration curve ranging from 1 to 63,000 pg/lL. The recovery of estrogens was 84% and it was assessed by adding 8.96 lg of 7-dehydrocholesterol (2.24 g/L) to each sample. The reported estrogen levels were corrected for this loss. 2.4. Statistical analyses To account for the non-independence of our data due to the repeated measurements performed at each site (spatio-temporal pseudoreplications), we used mixed effect models including sampling session and sites as random effects. The significance of the fixed effects was assessed using likelihood ratio (LR) tests (Edwards, 1992). Comparison of the contamination levels among the estrogens was carried out by including estrogen type as a fixed factor. Posthoc analyses were performed by conducting similar analyses for each estrogen pairs and by adjusting significance levels with appropriate Bonferroni corrections. To investigate spatial variation patterns, we conducted mixed effect models on the global estrogenic potency of the sediments, calculated according to typical predicted E2 equivalencies for in vivo vitellogenin induction in rainbow trout in the following manner: 0.001E3:0.5E1:1E2:25EE2 (Johnson and Sumpter, 2001). Using a global estimation of the estrogenic potency rather than individual levels of steroid estrogen is more biologically meaningful, since steroid estrogens may act in additive ways (Brian et al., 2005; Thorpe et al., 2003). The mixed effect models of E2 equivalent concentrations included site as a fixed effect, and STW treatment type and the size of the population served by the nearest sewage treatment work as main factors. For illustrative purposes, we also carried out the simple linear regression of the average E2 concentrations in relation to human population sizes. Note, however, that this analysis was only used here as a complementary approach and essentially for illustrative purposes, since working on average values rather than on raw values is likely to produce loss of statistical power due to loss of information regarding intra-group variation. Finally, to determine whether the detected patterns result from one or several estrogens, we carried out separate linear mixed models on each of the measured estrogens. Significance of population size was determined as above with LR tests with appropriate Bonferroni corrections. All statistical analyses were conducted using R 2.9.2. 3. Results 3.1. General patterns of steroid estrogens contamination Inspection of the statistical distributions revealed suspiciously high levels of steroid estrogens in the samples collected off Penco during the first sampling session (see Supplementary material). For EE2, distances to the mean of the detected concentrations were up to 51 times higher than the standard deviations. Such abnormally high levels were not observed in the samples collected at the same location during the second sampling trip. This suggests that, by chance, we might have collected our samples directly from the sewage discharge during the first sampling session performed

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at this site. To comply with the application conditions of the statistical tests that we subsequently carried out, we excluded from our analyses the corresponding data. They are therefore not included in the detected contamination patterns we present. In every sample, we identified the presence of all four estrogens (See Supplementary material). Nevertheless, significant differences were observed in their contamination levels (LR = 95.243, df = 3, P < 0.001). The detected concentrations of E1 and E2 ranged from 0.06 to 4.61 ng/g dry weight (dw) and from 0.06 to 16.81 ng/g dw, respectively. According to our post hoc analyses, E1 and E2 levels do not differ significantly between each other (LR = 0.34, df = 1, P = 0.56). However, both tend to be lower than E3 concentrations (LR = 14.51 and 15.13, df = 1, P < 0.01 after Bonferroni corrections, Fig. 2), the later varying between 0.01 and 53.21 ng/g dw. With concentrations ranging from 4.18 to 48.14 ng/g dw, EE2 was overall the most prevalent estrogen detected (LR = 61.43, 58.81 and 26.46, df = 1, P < 0.001 after Bonferroni corrections, see Fig. 2). In fact, it was found to be more concentrated than any other estrogen at all but one station; and this regardless of whether or not the nearest sewage treatment plant performed biological treatment (see Table 1 and Fig. 2). On average, the levels of EE2 were 38– 67 times higher than those found for natural estrogens. At the site where E3 supplanted EE2 in terms of concentration (Talcahuano, Fig. 2), levels of the synthetic hormone were similar to those reported at other sampling sites; concentrations were high and fell within the range found at other stations (i.e. 10.85–48.14 ng/g dw for Talcahuano and 4.18–46.45 ng/g dw for the other stations). At this site, which is also impacted by wastewaters from fishmeal companies (Table 1), E3 levels appeared exceptionally high (ranges

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of E3: 10.26–53.21 ng/g dw for Talcahuano and 0.01–8.47 ng/g dw for the other stations). 3.2. Spatial analysis of the steroid estrogen contamination Elevated E2 equivalent concentrations were calculated for all stations (see Supplementary material). They ranged from 104.59 to 1203.97 ng/g dw and exhibited significant spatial variation (site effect: LR = 20.3, df = 8, P < 0.01). While we failed to demonstrate an effect of the type of treatment performed by the STWs (Treatment type: LR = 0.15, df = 1, P = 0.70) on the E2 equivalent concentrations, both our mixed effect analysis and the linear regression revealed a significant influence of the size of the human population served by the nearest treatment plant (Mixed effect model: Population size effect: LR = 6.19, df = 1, P = 0.01; linear regression: R2 = 0.64, F1,7 = 12.28, P < 0.01). Note that even if the detected relationship seemed strongly influenced by the large estrogenic loads found in the samples collected at Concepción (i.e. the largest population in Fig. 3), analyses performed after excluding these data from the mixed effect analyses still tend to support the positive relationship previously detected (Mixed effect model: Population size effect: LR = 2.77, df = 1, P = 0.09) and still indicate a positive correlation between estrogenic loads and size of the human population (r = 0.53, df = 6, unilateral P = 0.09). The fact that only borderline significances were detected in such a condition is likely due to the low power of the analyses (Power of the correlation analysis: 37%). Our post hoc analyses, performed on E1, E2, E3 and EE2 separately, only detected a significant influence of population size on sediment concentrations for EE2 (Population size ef-

Fig. 2. Mean ± SD concentrations for E1, E2, E3, EE2 and for the EE2 equivalent concentrations. Results from the first sampling session are in dark grey and those for the second sampling trip are in light grey.

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Fig. 3. Regression between the average E2 equivalent concentrations and the size of the population served by the nearest sewage plants.

fect: LR = 6.32, df = 1, P < 0.05 after Bonferroni correction for EE2, while LR = 0.006–0.153, df = 1, P > 0.05 for the natural estrogens).

4. Discussion 4.1. Estrogenic contamination profile By demonstrating the presence of steroid estrogens in all the sediment samples, our study indicates that estrogenic contamination is widespread along central-southern Chile’s coast. Interestingly, we detected very clear and consistent pollution trends. E1 and E2 concentrations were within the same order of magnitude and relatively low at all the stations. E3 was always present at higher levels and was, consequently, the most abundant natural estrogen. In most cases, it is the synthetic hormone EE2 that displayed the highest concentrations. This type of contamination profile differs in several aspects from the patterns generally reported in freshwater systems impacted by human sewage. For instance, while the detected concentrations of the natural estrogens are within the range described by other monitoring studies (Braga et al., 2005b; Isobe et al., 2006; Labadie et al., 2007; Lopéz de Alda et al., 2002; Peck et al., 2004; Robinson et al., 2009; Ternes et al., 2002; Vigano et al., 2008; Williams et al., 2003), E1 was not more abundant than E2, as is usually reported for sewage effluents, river waters and sediments (Isobe et al., 2006; Baronti et al., 2000; D’Ascenzo et al., 2003; Williams et al., 2003; Labadie and Budzinski, 2005; Labadie et al., 2007; Peck et al., 2004; Ternes et al., 2002; Labadie and Hill, 2007). It remains uncertain, however, if such a trend equally applies to coastal sediments. In fact, other monitoring studies along the Pacific coasts also failed to demonstrate higher levels of E1 compared to E2 in sediments (Braga et al., 2005b; Schlenk et al., 2005). This suggests that degradation rates, sorption properties of the estrogens or conversion of E2 and E1 (Baronti et al., 2000) might vary according to local physico-chemical properties and confer specific contamination profiles to some aquatic systems. This is also suggested by the relatively high levels of EE2 that were detected. This compound was actually abundant and present at concentrations higher than E1 and E2 in all of our samples. While high levels of this synthetic hormone have been sporadically

reported in sediments (Petrovic et al., 2002a; Lopéz de Alda et al., 2002; Pojana et al., 2007), it is seldom detected or is detected at relatively low concentrations in aquatic environments (for review see Streck, 2009). Besides, it is usually present at much lower levels than natural estrogens. Based solely on contraceptive habits, we expected the contamination by EE2 to be lower than those usually reported in industrialized western countries. Indeed, because Chilean women are only moderate users of oral contraceptives, the average per head excretion of the synthetic estrogen EE2 is estimated to be about 20% lower in central-southern Chile than in western Europe (Bertin et al., 2009). The EE2 levels that we detected cannot be attributed to large human populations since the Chilean STWs serve, in contrast, a relatively small numbers of residents (Table 1). Altogether, this suggests that the detected EE2 levels reflect the propensity of this synthetic compound to accumulate in the studied sediments. Coastal areas, and central-southern Chile in particular, may offer favorable conditions for such a phenomenon to occur both by promoting the sorption of EE2 to sediments and by limiting its degradation. Indeed, empirical evidence indicates that EE2 sorption is enhanced by salinity and high organic carbon contents (Robinson et al., 2009; Bowman et al., 2002). Besides, this synthetic compound is extremely persistent under hypoxic and dark conditions (Czajka and Londry, 2006), conditions frequently present in sub-surface sediments, and reports indicate a lack of anaerobic degradation of EE2 over very long incubation periods (>3 years, Czajka and Londry, 2006). The coasts of the eastern Pacific hold one of the three most important oxygen minimum zones (OMZ) of the global ocean (Levin, 2003). On the continental shelf off central-south Chile, the presence of the OMZ generates long periods of hypoxic conditions over the sediments (Farias et al., 2004) and it is a critical factor in modulating the benthic community structure (Quiroga et al., 2005). The upper limit of the OMZ at about 36°S fluctuates between 80 and 280 m depth (Silva and Neshyba, 1979), although during periods of active upwelling (October–March) it can reach 20 m deep (Brandhorst, 1971). For instance, Farias et al. (2000) estimated that in Concepción Bay (36°400 S; 73°010 W; Tome, Penco, Talcahuano; Fig. 1), during, at least, 57% of the year, the bottom of the bay corresponded to a sub-oxic or anoxic environment. EE2 might be of high concern in environments like those with typical marine salinities and where organic content is high and oxygenation is low (Seguel et al., 2001; Farias et al., 1996; Farías, 2003; Farías and Salamanca, 1990). Interestingly, this hypothesis is also supported by a monitoring study conducted in the coastal lagoon of Venice where EE2 was the only estrogen steroid detected at relevant concentrations in sediments (Pojana et al., 2007). Physico-chemical properties of the sediments like total organic carbon, swelling clay content, state of flocculation, particle size, etc., may also affect sorption and desorption capacities of steroid estrogens (Duong et al., 2010; Zhou et al., 2007; Shareef et al., 2006). Further studies are thus necessary to evaluate whether sediment characteristics off the coast of central-southern Chile can explain the high levels of EE2 that we detected. 4.2. Spatial variation and origin of the estrogenic contamination While the omnipresence of steroid estrogens in the studied area raises concerns for the entire coast of central-southern Chile, our results indicate that some locations suffer higher estrogenic pollution than others and might, therefore, be more at risk. Contrary to our expectations, the highest estrogenicities were not observed in areas receiving discharges from STWs performing primary treatment only. In fact, our analyses failed to evidence a significant effect of the type of sewage treatment on estrogenic contamination. At first glance, this result might seem surprising since numerous studies have demonstrated that the removal efficiency of steroid

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estrogens is significantly improved by biological treatment (Khanal et al., 2006; Lee and Liu, 2002; Ternes et al., 1999). According to the meta-analysis of Johnson et al. (2007), the average removal efficiencies of activated sludge plants reach 78%, 91% and 76%, for E1, E2 and EE2, respectively; while removals of steroid estrogens during primary treatment are essentially nonexistent (Ternes et al., 1999; Svenson et al., 2003; Andersen et al., 2003; Johnson et al., 2005; Servos et al., 2005; Braga et al., 2005a). Our inability to demonstrate the influence of sewage treatment type on the estrogenic contamination, however, might simply reflect the lack of power of our analyses that used relatively small sample sizes (N = 5 and 3, respectively, for STWs performing biological treatment or not). In contrast, our analyses suggest a human contribution to the steroid estrogen contamination, which may explain a large part of the detected spatial variation. Of course, this result does not preclude the contribution of other contamination sources. Fishmeal waste, for instance, is likely to contribute to the exceptionally high levels of natural estrogens that we detected in Talcahuano, since this area also receives waste from fishmeal companies that process several hundred thousand tons of fish landings per year (Sernapesca, 2008; Afonso and Borquez, 2002; Landes, 2006).

5. Conclusions The present study indicates that releases of human sewage in the marine environment might result in significant sediment contamination by steroid estrogens. The high salinity and low oxygenation of coastal sediments may promote in particular the sorption of EE2 to sediments and limit its degradation. The impact of EE2 on marine biota remains to be explored. Theoretical and empirical studies indicate that while steroid estrogens in sediments are of direct concern for benthic species (Rempel et al., 2008; Dussault et al., 2009; Liebig et al., 2005; Langston et al., 2007; Lai et al., 2002), they may affect a broad range of species due to trophic transfer (Martinrobichaud et al., 1994; Lai et al., 2002). In conclusion, even though more empirical studies are needed, current knowledge indicates that contamination of marine sediments by steroid estrogens should not be neglected, particularly in areas which host edible fish and seafood, as this may turn out to be a significant health issue for humans.

Disclosure We assure that the ms is original work, which has not been previously published in whole or in part and that it is not under consideration for publication elsewhere. We declare that none of us has conflict of interest including any financial, personal or other relationships with other people or organizations within 3 years of beginning the work submitted that could inappropriately influence their work. All the authors have materially participated in the article. AB was involved in the setting up of the study design, in the analysis and interpretation of the data and she wrote the manuscript. PI was in charge of the data collection and participated in the writing of the manuscript. RQ was involved in the setting up of the study design, in the interpretation of the results and participated in the writing of the paper. All authors have approved the final version of the article.

Role of the funding source Celulosa Arauco and Constitución S.A, the study’s sponsor did not have any role in study design, in the collection, analysis and

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interpretation of data, nor in the writing and in the decision to submit the paper for publication. Acknowledgements This research is part of the Programa de Investigación Marina de Excelencia (PIMEX-Nueva Aldea) of the Faculty of Natural and Oceanographic Sciences of the University of Concepción, funded by Celulosa Arauco and Constitución S.A. The authors acknowledge Mauricio Mardanes and Eduardo Mora for their help in the field. We thank Silvio Pantoja and Rodrigo Castro as well for performing the chemical analyses of the sediment samples. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.marpolbul.2011.04.002. References Aerni, H.R., Kobler, B., Rutishauser, B.V., Wettstein, F.E., Fischer, R., Giger, W., Hungerbuhler, A., Marazuela, M.D., Peter, A., Schonenberger, R., Vogeli, A.C., Suter, M.J.F., Eggen, R.I.L., 2004. Combined biological and chemical assessment of estrogenic activities in wastewater treatment plant effluents. Anal. Bioanal. Chem. 378, 688–696. Afonso, M.D., Borquez, R., 2002. Review of the treatment of seafood processing wastewaters and recovery of proteins therein by membrane separation processes – prospects of the ultrafiltration of wastewaters from the fish meal industry. Desalination 142, 29–45. Ahel, M., Giger, W., Schaffner, C., 1994. Behaviour of alkyphenol polyethoxylate surfactants in the aquatic environment – II. Occurence and transformation in rivers. Water Res. 28, 1143–1152. Andersen, H., Siegrist, H., Halling-Sorensen, B., Ternes, T.A., 2003. Fate of estrogens in a municipal sewage treatment plant. Environ. Sci. Technol. 37, 4021–4026. Atkinson, S., Atkinson, M.J., Tarrant, A.M., 2003. Estrogens from sewage in coastal marine environments. Environ. Health Perspect. 111, 531–535. Baronti, C., Curini, R., D’Ascenzo, G., Di Corcia, A., Gentili, A., Samperi, R., 2000. Monitoring natural and synthetic estrogens at activated sludge sewage treatment plants and in a receiving river water. Environ. Sci. Technol. 34, 5059–5066. Beck, I.C., Bruhn, R., Gandrass, J., Ruck, W., 2005. Liquid chromatography–tandem mass spectrometry analysis of estrogenic compounds in coastal surface water of the Baltic Sea. J. Chromatogr. A 1090, 98–106. Bertin, A., Inostroza, P.A., Quiñones, R.A., 2009. A theoretical estimation of the concentration of steroid estrogens in effluents released from municipal sewage treatment plants into aquatic ecosystems of central-southern Chile. Sci. Total Environ. 407, 4965–4971. Bowman, J.C., Zhou, J.L., Readman, J.W., 2002. Sediment–water interactions of natural oestrogens under estuarine conditions. Mar. Chem. 77, 263–276. Braga, O., Smythe, G.A., Schafer, A.I., Feitz, A.J., 2005a. Fate of steroid estrogens in Australian inland and coastal wastewater treatment plants. Environ. Sci. Technol. 39, 3351–3358. Braga, O., Smythe, G.A., Schafer, A.I., Feitz, A.J., 2005b. Steroid estrogens in ocean sediments. Chemosphere 61, 827–833. Braga, O., Smythe, G.A., Schafer, A.I., Feltz, A.J., 2005c. Steroid estrogens in primary and tertiary wastewater treatment plants. Water Sci. Technol. 52, 273–278. Brandhorst, W., 1971. Condiciones oceanográficas estivales frente a la costa de Chile. Rev. Biol. Mar. 14, 45–84. Brian, J.V., Harris, C.A., Scholze, M., Backhaus, T., Booy, P., Lamoree, M., Pojana, G., Jonkers, N., Runnalls, T., Bonfa, A., Marcomini, A., Sumpter, J.P., 2005. Accurate prediction of the response of freshwater fish to a mixture of estrogenic chemicals. Environ. Health Perspect. 113, 721–728. Caliman, F.A., Gavrilescu, M., 2009. Pharmaceuticals, personal care products and endocrine disrupting agents in the environment – a review. Clean: Soil, Air, Water 37, 277–303. Carballo, M., Aguayo, S., de la Torre, A., Munoz, M.J., 2005. Plasma vitellogenin levels and gonadal morphology of wild carp (Cyprinus carpio L.) in a receiving rivers downstream of Sewage Treatment Plants. Sci. Total Environ. 341, 71–79. Cargouet, M., Perdiz, D., Levi, Y., 2007. Evaluation of the estrogenic potential of river and treated waters in the Paris area (France) using in vivo and in vitro assays. Ecotoxicol. Environ. Saf. 67, 149–156. Chen, C.Y., Wen, T.Y., Wang, G.S., Cheng, H.W., Lin, Y.H., Lien, G.W., 2007. Determining estrogenic steroids in Taipei waters and removal in drinking water treatment using high-flow solid-phase extraction and liquid chromatography/tandem mass spectrometry. Sci. Total Environ. 378, 352–365. Czajka, C.P., Londry, K.L., 2006. Anaerobic biotransformation of estrogens. Sci. Total Environ. 367, 932–941. D’Ascenzo, G., Di Corcia, A., Gentili, A., Mancini, R., Mastropasqua, R., Nazzari, M., Samperi, R., 2003. Fate of natural estrogen conjugates in municipal sewage transport and treatment facilities. Sci. Total Environ. 302, 199–209.

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