Occurrence of selected endocrine disrupting compounds in Iberian coastal areas and assessment of the environmental risk

Accepted Manuscript Occurrence of selected endocrine disrupting compounds in Iberian coastal areas and assessment of the environmental risk N. Salgueiro-González, J.A. Campillo, L. Viñas, R. Beiras, P. López-Mahía, S. Muniategui-Lorenzo PII:

S0269-7491(18)35092-9

DOI:

https://doi.org/10.1016/j.envpol.2019.03.107

Reference:

ENPO 12379

To appear in:

Environmental Pollution

Received Date: 18 December 2018 Revised Date:

11 March 2019

Accepted Date: 25 March 2019

Please cite this article as: Salgueiro-González, N., Campillo, J.A., Viñas, L., Beiras, R., López-Mahía, P., Muniategui-Lorenzo, S., Occurrence of selected endocrine disrupting compounds in Iberian coastal areas and assessment of the environmental risk, Environmental Pollution (2019), doi: https:// doi.org/10.1016/j.envpol.2019.03.107. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphical abstract

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Endrocrine Disrupting Compounds (EDCs)

CONTAMINATED WATER

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OCCURRENCE OF SELECTED ENDOCRINE DISRUPTING COMPOUNDS

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IN

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ENVIRONMENTAL RISK

IBERIAN

COASTAL

AREAS

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ASSESSMENT

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N. Salgueiro-González*a, J. A. Campillob, L. Viñasc, R. Beirasd, P. López-Mahíaa; S.

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Muniategui-Lorenzoa

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a

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Centro de Investigaciones Científicas Avanzadas (CICA), Facultade de Ciencias, Universidade

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da Coruña, 15071 A Coruña, Galicia, Spain.

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b

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30740 San Pedro del Pinatar, Murcia, Spain.

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c

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50, 36390 Vigo, Spain

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Marcosende, 36200, Vigo, Galicia, Spain.

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Instituto Español de Oceanografía, Centro Oceanográfico de Murcia, Apdo. 22, C/Varadero 1,

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Instituto Español de Oceanografía, IEO, Centro Oceanográfico de Vigo, Subida a Radio Faro,

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Grupo Química Analítica Aplicada, Instituto Universitario de Medio Ambiente (IUMA),

Departamento de Ecoloxía e Bioloxía Animal, Universidade de Vigo, Campus Lagoas-

*Corresponding author: [email protected] (N. Salgueiro-Gonzalez)

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ACCEPTED MANUSCRIPT ABSTRACT

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The spatial and temporal distribution of selected endocrine disrupting compounds (4-

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tert-octylphenol, 4-n-octylphenol, 4-n-nonylphenol, nonylphenol, and bisphenol A) in

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two coastal areas of the Iberian Peninsula (Ria de Vigo and Mar Menor lagoon) were

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evaluated for the first time. Seawater and sediment samples collected during spring and

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autumn of 2015 were analysed using greener extraction techniques and liquid

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chromatography-tandem mass spectrometry. The presence of branched isomers (4-tert-

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octylphenol and nonylphenol) and bisphenol A in almost all seawater and sediment

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samples demonstrated their importance as pollutants in the frame of water policy, while

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no concentrations of linear isomers (4-n-octylphenol and 4-n-nonylphenol) were found.

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Higher seawater levels were observed in Mar Menor lagoon, especially in spring,

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associated with wastewater treatment plant effluents and nautical, agricultural and

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industrial activities. Similar sediment concentrations were measured in both studied

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areas, being nonylphenol levels five times higher than those measured for the other

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EDCs. Experimental sediment–water partition coefficients shown a moderate sorption

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of target compounds to sediments. Risk quotients for water compartment evidenced a

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moderate risk posed by nonylphenol, considering the worst-case scenario. For

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sediments, moderate risk related to 4-tert-octylphenol and high risk to nonylphenol

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were estimated.

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Capsule: Occurrence, distribution and environmental impact of selected endocrine disrupting

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compounds in marine environment

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Keywords: alkylphenols, bisphenol A, plastic additives, marine environment, risk quotient

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1. INTRODUCTION

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The global plastic manufacture has rapidly developed over the past years, with a

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total amount of 280 million tonnes in 2016 (Plastics Europe, 2018). The impact of

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(micro)plastics and their additives to the aquatic environment has aroused special

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concern because of the marine litter found in open oceans. Some of these additives, such

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as 4-alkylphenols (4-APs) have gained particular attention due to their toxicity and their

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behaviour as endocrine disrupting compounds (EDCs). These compounds are the main

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degradation products of the non-ionic surfactants alkylphenol ethoxylates (APEs),

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extensively used for cleaning formulations (Acir and Guenther, 2018). It is well-known

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that 4-APs affect the hormonal system of humans and wildlife at low concentrations and

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consequently, they have been considered as priority substances within water policy in

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the frame of Directive 2013/39/EU (Directive-2013/39, 2013).

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Another remarkable EDC is bisphenol A (BPA), employed in the manufacture of

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polycarbonate plastic and epoxy resins, and also as a component of flame retardants,

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adhesives and electronic circuits (Gallart-Ayala et al., 2010). European Union has not

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considered BPA as a priority substance in waters. However, BPA has been identified as

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a substance of very high concern (SVHCs) because of its reproductive toxicity and it

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has been recently included in the “Candidate List for eventual inclusion in Annex XIV

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to REACH” (European Chemical Agency (ECHA), 2017). In fact, different

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ecotoxicological studies have demonstrated its effects in organisms, such as sexual

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maturation, altered development, and tissue organisation of mammary glands at trace

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concentrations (Flint et al., 2012; Tato et al., 2018; Wei et al., 2011). Consequently, the

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levels and behaviour of BPA in the environment should be investigated in order to

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(re)evaluate the controversial situation generated around this EDC.

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ACCEPTED MANUSCRIPT A great number of authors have reported the presence of 4-APs and BPA in

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surface waters, primarily in association with wastewater treatment plants (WWTP) and

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industrial discharges (Vilela et al., 2018; Wilkinson et al., 2018). Nevertheless, their

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behaviour in the marine ecosystem remains partially unknown to date. Few research

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studies have investigated the impact of branched 4-APs and BPA (and to a lesser extent

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linear 4-APs) in seawaters and sediments from coastal areas affected by large inputs

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derived from urban, industrial, agricultural activities (see Table S1 and Table S2 of

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Supplementary Material, SM). Thus, the investigation of these EDCs in the marine

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environment is of great concern in order to avoid and control chemical pollution,

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preserve the ecosystem, and protect human health.

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Two representative Spanish coastal areas were covered in this study due to their

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natural, social, and economic importance in the Iberian Peninsula. Ria de Vigo is one of

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the most important Galician estuaries known for their industrial, fishing, shipping and

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aquaculture activities, such as mussel and oyster farming. In fact, shellfish production in

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Galicia ranks first in Spain, with more than 90% of the total production (Sánchez-Marín

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and Beiras, 2008). On the other hand, Mar Menor is a coastal lagoon located in the

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south-eastern of Spain and connected to the Mediterranean Sea, relevant in terms of

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agricultural, touristic, industrial and urban/recreational activities. The presence and

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distribution of trace metals (Quelle et al., 2011) and organic pollutants (León et al.,

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2017; Moreno-González et al., 2013), such as polycyclic aromatic hydrocarbons (PAHs)

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(Viñas et al., 2009), pharmaceutical compounds (Moreno-González et al., 2015) and

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non-ionic surfactants (Traverso-Soto et al., 2015) in one or the other area have already

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been investigated. Only one previous research studied the occurrence and spatial

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distribution of target compounds in Galician and Cantabrian estuaries (Salgueiro-

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González et al., 2015), including some sampling points in Ria de Vigo, but it focused on

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ACCEPTED MANUSCRIPT the analysis of seawater samples (one sampling campaign in 2012) and did not consider

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sediment analysis. Since sorption processes are a key factor for the reactivity, mobility,

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persistence and bioavailability of EDCs in natural waters, there is a need to understand

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their interactions with sediments. To the best of our knowledge, this is the first

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integrative study based on 4-APs and BPA in these coastal areas, allowing the

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comparison between two marine ecosystems with different anthropogenic pressures and

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contaminant inputs.

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In the light of the above-mentioned concerns, the aims of this paper are: (1) to

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comprehensively characterise the occurrence, spatial distribution and seasonal

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variations of selected EDCs (4-APs and BPA) in seawaters and sediments from two

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relevant coastal areas in Spain (Ria de Vigo and the Mar Menor lagoon); (2) to identify

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possible sources of anthropogenic contamination by target compounds in the studied

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areas in order to evaluate and control chemical pollution and finally, (3) to assess the

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environmental risk that these compounds pose to the marine environment in those areas

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in order to be able to preserve the aquatic ecosystem and guarantee public safety.

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2. MATERIAL AND METHODS

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2.1. Study area and sampling procedure

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As shown in Fig. 1, two coastal areas subjected to different physical, chemical

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and hydrodynamic conditions and to diverse predominant human activities were

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considered: Ria de Vigo (Fig. 1A) and Mar Menor lagoon (Fig. 1B). Ria de Vigo (Rías

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Baixas, NW Spain) is a wedge-shaped estuary orientated in a north-east to south-west

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direction, with a length of 35 km. Maximum width occurs at the mouth (8 Km), where

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the Cies Islands are located, partially closing off the estuary. The width is gradually

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reduced to 1 km at Rande Strait. There are a number of small, dispersed population

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exceeds 300,000 inhabitants and enjoys a considerable industrial development and a

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significant port operation. The Lagares wastewater treatment plant (WWTP) outfall is

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situated in the central area of Ria de Vigo (Fig. 1A) and is the largest in this area,

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although other WWTP effluents are also discharged to this estuary (i.e. Cangas, Moaña,

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Redondela, Comboa-Sotomayor). All this is in addition to geographic and

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oceanographic characteristics that do not allow a good level of water exchange with the

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ocean, which could lead to an accumulation of certain contaminants within the estuary

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(Fernández et al., 2016).

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The Mar Menor (Murcia, SE Spain) is a hypersaline lagoon (42-47 psu) with a

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mean depth of 4.5 m and a maximum depth of 6.6 m, being one of the largest coastal

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lagoons in the Mediterranean Sea. It is isolated from the sea by a 24 Km long sand bar

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(called La Manga) that is only crossed by five channels, causing seasonal fluctuations

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of temperature and salinity higher than those detected in the former. It is located close to

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the Campo de Cartagena area, characterized by intensive agriculture, recreational

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activities and a sporadic torrential rainfall regime. It also receives wastewater effluents

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from Los Alcázares WWTP (Fig. 1B) and brackish water effluents (salinity varies from

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4 to 11 psu) (García-Pintado et al., 2007). The impact of tourism activities has also

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increased over the past years, with dense urban and tourist developments on the shores

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of the Mar Menor lagoon, and the proliferation of coastal settlements and ports.

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Water and sediment samples were collected along both estuaries during spring

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(April) and autumn (September-October) of 2015, in order to evaluate the spatial and

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temporal distribution of EDCs. Sampling points (Fig. 1) were chosen according to

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previous studies in these areas and to the proximity of possible terrestrial and marine

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sources of pollution for the target compounds. In spring, five sampling points were

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ACCEPTED MANUSCRIPT considered in Ria de Vigo (RV1-RV5) and the Mar Menor lagoon (MM1-MM5). The

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external sampling stations located in the Atlantic Ocean (outer side of the Cies Islands)

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and Mediterranean Sea were considered as reference points for respectively Ria de Vigo

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(RVR) and Mar Menor lagoon (MMREF), where no (or low) concentrations of EDCs

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are expected. In autumn, one additional reference station was added in both areas,

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taking into account the levels measured during the spring campaign (RVR2, MMRF2).

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Moreover, in the case of the Mar Menor lagoon two additional sampling points were

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also considered in autumn in order to complete the spatial distribution of the lagoon

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(MM6 and MM7).

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Seawater samples were collected in surface and deep seawater layers (at 1 m

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above the seafloor) in Ria de Vigo during low tide period in order to achieve a better

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identification of main terrestrial pollution sources. Only surface seawaters (1-15 cm)

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were taken in the Mar Menor lagoon. All samples were collected in 1L amber glass

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bottles using a glass pitcher and stored at 10ºC until arrival at the laboratory, and then at

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-20 ºC until further analysis. Surface sediment samples (0-1.5 cm depth) were collected

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at the same time as waters during the spring and autumn campaigns in both areas.

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Sampling was carried out using a Van-Veen Grab in the Mar Menor lagoon and using a

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box corer in Ria de Vigo, taking 4-5 samples at each station and pooling them in order

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to get a more representative data. Next, the sediment samples were well-homogenized

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and deposited on aluminium trays previously rinsed with hexane. Sediments were

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frozen at -20ºC, then freeze-dried, homogenised and sieved through a 2 mm mesh

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before the analysis. Details about sediment properties (i.e. total organic carbon and

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granulometry) are reported in SM.

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2.2. Chemicals

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Information about chemical and suppliers is provided in the SM. 7

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2.3. Analytical methodology: QA/QC considerations Seawater and sediment samples were analysed using greener methods previously

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published by Salgueiro-González et al. (Salgueiro-González et al., 2014, 2012a), based

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on a dispersive liquid-liquid microextraction (DLLME) and a selective pressurized

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liquid extraction (SPLE), respectively. In both cases, the determination was performed

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by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Plastic material

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and detergents were discarded in sampling and sample treatment to avoid blank

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problems (Salgueiro-González et al., 2012b). Analytical methods were validated in

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terms of linear range, accuracy, precision and selectivity. Recoveries varied from 91 to

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101% (RSD<9%) for seawaters and from 97 to 100% (RSD<6%) for sediment samples.

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Method quantitation limits (MQL) ranged between 0.005 and 0.02 µg L−1 and between

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0.1 and 4.0 µg kg−1 dw for respectively seawaters and sediments. Instrumental and

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procedural blanks were included in each analytical run to check for eventual

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contaminations. Further details about the analytical methods (i.e. extraction, clean-up

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and determination) and their validation step can be found in SM.

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2.4. Risk assessment

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Risk quotients (RQs) were estimated for evaluating the risk posed by the three

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measured EDCs (4-tOP, NP and BPA) on the aquatic ecosystem, considering both

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environmental compartments (i.e. waters and sediments). RQ is normally defined as the

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ratio of measured environmental concentration (MEC) to the predicted no-effect

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concentration (PNEC). However, MEC values can be represented by the mean or

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maximum detected concentrations, depending on the pollution scenario (Palma et al.,

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2014). Considering a general scenario (RQm), MEC was calculated as the geometric

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mean of all the samples for each region in both sampling campaigns. For samples in

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which EDC concentration was lower than MQL, the half of its MQL was taking into

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account, according to the Directive 2009/90/EC (Directive-2009/90, 2009). When the

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worst-case scenario (RQex) was investigated, MEC values were the maximum detected

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concentrations in both sampling campaigns. PNEC values are usually derived from the lowest toxicity value (i.e. lowest

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short-term LC50 or long-term no-observed effect concentration (NOEC) value)

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observed for the most sensitive species, and by application of an assessment factor (AF)

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(generally from 10 to 1000 according to the available data) (European-Commission,

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2003). In our study, the PNEC in water compartment (PNECwater) was determined

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considering NOEC values for three species across three trophic levels (Table 1) and

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therefore, an AF of 10 was applied (European-Commission, 2003). Microalgae

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(Isochrysis galbana), sea-urchin (Paracentrous lividus) and fish (Oncrhynchus mykiss)

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were selected as representative species of respectively primary producers, primary

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consumers and secondary consumers in marine environment. For sediments, PNECsed

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were estimated by the equilibrium partitioning method (European-Commission, 2003),

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which is detailed in SM. Using an accepted criterion for RQ, three different levels of

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concern can be considered: low risk (RQ<0.1), moderate risk (0.1≤RQ<1) or high risk

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(RQ≥1) (Blair et al., 2013; Palma et al., 2014).

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3. RESULTS AND DISCUSSION

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3.1. Distribution of 4-APs and BPA in seawaters

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In order to know the occurrence, spatial and temporal distribution of 4-APs and

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BPA in water samples, the levels of the target compounds measured in each sampling

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point, location and campaign are discussed in the following lines. Furthermore, a

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comparison between different regions with different chemical characteristics and inputs

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is also included. 3.1.1. Ria de Vigo

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The measured concentrations of 4-APs and BPA in Ria de Vigo are shown in

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Fig. 2A. As it can be seen, the concentrations of linear 4-APs isomers (4-n-OP and 4-n-

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NP) were lower than MQL in all samples which can be explained due to the lower

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industrial applications of these compounds as well as their faster degradation (Pothitou

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and Voutsa, 2008). Regarding branched 4-APs (4-tOP and NP) and BPA, differences

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can be observed between both seasons. In spring, none of the compounds was measured

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(
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The number of positive samples for 4-tOP, NP and BPA were respectively 18%, 91%

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and 41%. For 4-tOP, concentrations ranging from <0.008 and 0.011 µg L-1 (RV1) were

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found. In all cases the presence of 4-tOP was detected in surface waters from the inner

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part of this Ria, probably associated with the inputs of the Verdugo and Alvedosa rivers

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and the WWTP discharges from Redondela (30,000 inhabitants). NP was measured in

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almost all sampling points at levels between 0.031 and 0.056 µg L-1 (RV5), which can

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be explained by the influence of harbours, urban locations and WWTP distributed along

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the Ria de Vigo. Furthermore, the similar concentrations found in surface and bottom

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waters indicated its homogenate distribution in water bodies. Regarding BPA, levels

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ranged from <0.020 and 0.054 µg L-1, with the highest values of 0.34 µg L-1 (RV1) and

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0.27 µg L-1 (RV2) related to WWTP (Redondela) and industrial discharges. The fact that

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higher concentrations were measured in surface waters was explained by freshwater

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discharges.

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In autumn, no concentration of any compound was measured in the first

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reference point (RVR), whereas significant levels of NP (0.068 µg L-1) and BPA (0.035 10

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dumping favoured by the ocean currents and water inputs in this side of the Cies

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Islands. A decrease in the number of positive samples by NP (50%) could be observed

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whereas a remarkable increase was detected for 4-tOP (35%) and BPA (57%). In the

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case of 4-tOP, levels of 0.021 µg L-1 (RV3), 0.028 µg L-1 (RV5), and 0.041 µg L-1 (RV4)

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were found in surface seawaters, which were two or four times higher than those

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measured in spring. The harbours located in Vigo and Cangas and the navigation

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activity associated with them were considered the main contamination sources in RV3

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and RV4, probably because of detergents and cleaning products used in ship

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maintenance (Jonkers et al., 2010). In the sampling site where the maximum

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concentration was found in spring (RV1), 4-tOP was not detected in autumn maybe due

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to the lower contribution to Verdugo River in this sampling campaign. Beside the

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second reference point, NP and BPA were determined in the same points as 4-tOP

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(RV3, RV4 and RV5). Levels ranging from 0.037 and 0.088 µg L-1 for NP (RV5) and

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between 0.025 and 0.050 µg L-1 (RV3) for BPA were found. It should be noted that

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BPA was not detected in RV1 and RV2, where maximal concentrations were found in

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spring. This fact results from the high spatial and temporary variability of hydrophobic

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contaminants in coastal seawaters offered by a spot value. Consequently, integrated

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samples (sediment and biota) can be more representative than seawater for these

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substances, particularly when discontinuous inputs and discharges are present. In

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addition to harbours, WWTP located in Cangas (RV4) (26,000 inhabitants) and the

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direct urban and industrial waste discharges into the Ria de Vigo near Oia (3,000

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inhabitants) could be considered as sources of contamination by these compounds. In

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the majority of cases, similar concentrations of 4-tOP, NP and BPA were measured in

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surface and bottom waters. NP levels were lower than the maximum allowable

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ACCEPTED MANUSCRIPT concentration (MAC) value (2 µg L-1) set by Directive 2013/39/EU. No MAC value for

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4-tOP in waters is established by this legislation and measured concentrations were

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therefore compared with the annual average (AA) in seawaters (0.01 µg L-1), knowing

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the limitations of the comparison. In the case of a positive sample, 4-tOP levels

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exceeded the AA showing the needed for further research to obtain a reliable value and

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to control the chemical pollution.

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3.1.2. Mar Menor lagoon

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As in Ria de Vigo, no concentrations of linear alkylphenols (4-n-OP and 4-n-

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NP) could be measured in the Mar Menor seawater samples (detected at levels below

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MQL). Higher levels of 4-tOP, NP and BPA were measured in the Mar Menor lagoon

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(Fig. 2B) compared to those in Ria de Vigo, probably due to its low dilution capacity

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and high water residence time. In spring, remarkable percentages of positive samples for

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4-tOP (33%), NP (100%) and BPA (50%) were found. For 4-tOP, concentrations

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ranging between <0.008 and 0.010 µg L-1 (MM1 and MM2) were detected associated

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with the presence of a port in an urban area, such as in San Pedro del Pinatar (24,000

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inhabitants) and Los Alcázares (16,000 inhabitants). The presence of NP in the

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Mediterranean station (MMREF) at a concentration of 0.051 µg L-1 showed its ubiquity,

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probably related to San Pedro del Pinatar WWTP outfall. NP levels ranged from 0.031

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(MM1) to 0.11 µg L-1 (MM5); the highest concentration could be explained by the

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proximity to the area of Tomas Maestre Port and El Estacio channel, where the biggest

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yacht port is located. Regarding BPA, concentrations ranging between <0.020 and 0.48

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µg L-1 (MM2) were measured. This high value, probably associated with the influence

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exerted by Los Alcázares urban area and San Javier-Murcia airport, is more than ten

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times higher than other concentrations found in this area.

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However, a percentage of positive samples lower than in spring was obtained for NP

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(66%) and BPA (33%). Higher concentrations of 4-tOP were measured in autumn

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compared to spring, which ranged from <0.008 and 0.045 µg L-1 (MM7). This sampling

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point, not included in the spring campaign, is also located near the yacht port of Tomás

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Maestre. Another sampling site not included in the previous campaign (MM6) and

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located near a small harbour in the south of the lagoon showed an appreciable

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concentration of 4-tOP (0.028 µg L-1). Levels of NP ranging from 0.03 (MMRF2) to

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0.15 µg L-1 (MM7) were measured. The highest concentration of NP was measured in

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the same sampling points as 4-tOP which showed the common sources of pollution for

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these APs. Concerning BPA, values between 0.026 (MMR2) and 0.064 µg L-1 (MM3)

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were found, related to WWTP discharges of Los Alcázares. The highest concentration

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of BPA measured in MM2 in the spring season was minimised in autumn. As it can be

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seen, whereas the first reference sampling point showed NP concentration, the second

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one was positive in 4-tOP, NP and BPA concentrations, probably because of its

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proximity to La Manga and Canal del Estacio. In the case of Mar Menor, NP levels

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were lower than the MAC (2 µg L-1) value set by water legislation; however, almost all

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4-tOP measured concentrations exceeded the AA value (0.01 µg L-1) established by

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Directive 2013/39/EU. As in the case of Ria de Vigo, further research is needed to

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obtain a reliable value and to control the chemical pollution by this compound in this

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lagoon, if necessary.

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3.1.3. Comparison with other locations

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More than 85% of the works reported in SM (Table S1) investigated branched 4-

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APs and BPA, whereas less than 12% included linear 4-APs. As linear isomers were not

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found in this study, the comparison was limited to the other EDCs, although low levels

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of linear 4-APs were found in estuaries in Singapore (Basheer and Lee, 2004) and

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Brazil (Lisboa et al., 2013). Levels found in this study are in agreement with a previous study carried out in

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five Galician estuaries (Salgueiro-González et al., 2015). As far as we know, no levels

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of 4-APs and BPA in the Mar Menor lagoon have been reported yet. However, they

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were measured at similar levels in other coastal areas in Spain, such as the Cantabrian

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coast (Iparraguirre et al., 2012; Sánchez-Avila et al., 2013). Three Spanish cities located

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on the Mediterranean coast were investigated by Petrovic et al. (Petrovic et al., 2002),

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with the highest concentrations (more than ten times higher than those in our study)

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found in Barcelona (1.6 million inhabitants), in sampling points collected near the

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harbour and influenced by WWTP discharges and Besòs river contribution. Other

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European areas such as the Venice lagoon (Italy) (Pojana et al., 2007) and Thermaikos

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Gulf (Greece) (Arditsoglou and Voutsa, 2012) received the impact of these EDCs by

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industrial and WWTP outfalls. Lower levels for branched 4-APs were found on the

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Iberian coast, more specifically in Ria de Aveiro in Portugal (70,000 inhabitants),

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related to an effective dilution of the WWTP discharges by a submarine outfall into the

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Atlantic ocean (Jonkers et al., 2010). Concerning other regions around the world, higher

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concentrations (from two to ten times) were found in Singapore (Basheer and Lee,

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2004), Brazil (Lisboa et al., 2013) and China (Diao et al., 2017; Li et al., 2013; Xu et

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al., 2018), which can be explained considering the overpopulation, the high number of

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industries in these areas, and the different industrial regulations.

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3.2. Distribution of 4-APs and BPA in sediment samples

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Marine sediment collected in Ria de Vigo and Mar Menor were analysed in

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order to investigate the distribution of target EDCs in this environmental compartment.

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Moreover, a comparison with other studies that consider locations with different 14

ACCEPTED MANUSCRIPT characteristics and inputs was included. Linear 4-APs were not considered in the

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following discussion as they were detected at levels below MQL. As this distribution

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depends on sediment properties, among other factors, the correlations between

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measured concentrations and TOC and granulometry were evaluated and reported in

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SM.

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3.2.1. Spatial distribution in Ria de Vigo

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The concentrations measured in sediments from Ria of Vigo were shown in Fig.

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3A. In spring, more than 50% of samples were positive in terms of 4-tOP, NP and BPA

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while no compounds were found in the reference sampling point (RVR) as well as in

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RV2. Considering positive samples, the concentrations of 4-tOP ranging between 14.4

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(RV4) and 33.0 µg kg-1 dw (RV1) were measured, corresponding the maximum

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concentration to the highest level measured in seawater, near WWTP of Redondela.

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Higher levels of NP were determined and varied from 364 (RV4) to 518 µg kg-1 dw

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(RV3), which demonstrated the ubiquity of this pollutant and the wide-spread pollution

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inside the estuary during that season. For BPA, concentrations ranging between 6.9

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(RV5) and 25.8 µg kg-1 dw (RV4) were found. At the last sampling point (RV4), no

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water concentration of BPA was detected. The seawater analysis offers a spot

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information, which is subjected to high spatial and temporal variability; for this reason,

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the analysis of integrative samples (e.g. sediment, biota) is more environmentally

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representative than punctual seawater analysis.

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In autumn, a higher number of positive samples was observed (>85% for 4-tOP,

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NP and BPA). As occurs with seawater samples, no compounds were detected in

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reference point (RVR); however, concentrations of 16.4, 100 and 4.30 µg kg-1 dw for

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respectively 4-tOP, NP and BPA were found in the additional sampling point included

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in autumn (RVR2). In general, the concentrations of 4-tOP, NP and BPA were found to 15

ACCEPTED MANUSCRIPT 372

be lower in autumn than spring, with the exception of sampling point RV2, where

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higher levels were observed. The highest concentrations determined in autumn for each

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compound were measured in RV4, at levels of 24.3 (4-tOP), 245.0 (NP) and 27.9 (BPA)

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µg kg-1 dw. Although no EQS for branched 4-APs in sediments are set by water legislation,

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measured concentrations were compared with different tentative values found in

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literature. Limits of 3.4 µg kg-1 dw and 180 µg kg-1 dw in marine sediments for 4-tOP

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and NP respectively were adopted by the European criteria (European Union, 2005a)

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(European Union, 2005b). These values were exceeded in almost all positive samples

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and therefore, more data relating environmental concentrations and biological effects

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are needed to set reliable values and control chemical pollution by these EDCs for

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protecting the environment.

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3.2.2. Spatial distribution in Mar Menor lagoon

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EDC concentrations measured in the Mar Menor lagoon are illustrated in Fig.

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3B. Compared to Ria de Vigo, higher concentrations for 4-tOP, similar for NP and

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lower for BPA were found. These differences can be attributed to the variable

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characteristics observed in both locations, which play an important role in processes of

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sorption to sediments.

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In spring, 4-tOP, NP and BPA were found in all sampling points, including the

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reference sampling point (MMREF). Levels ranging from 9.9 (MM4) to 40.3 µg kg-1 dw

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and from 201 (MMREF) to 601 µg kg-1 dw were found for respectively 4-tOP and NP.

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Both maximum concentrations were measured in MM1 corresponding to the highest

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seawater levels, located close to San Pedro del Pinatar urban area and Lo Pagán port.

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Lower levels of BPA were measured along the coastal lagoon, ranging from 3.2

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(MMREF) to 12.0 µg kg-1 dw (MM5), because of the lower capability of this pollutant

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to be accumulated in sediments, compared to that of NP. In autumn, the number of positive samples was lower than in spring, with

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percentages of 88% for 4-tOP, 77% for NP and 66% for BPA. Whereas no

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concentration of any compound was observed in the initial reference sampling point

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(MMREF), the concentration of branched isomers (40.0 and 67.3 µg kg-1 dw for 4-tOP

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and NP, respectively) was measured in the additional reference sampling point

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(MMRF2), probably due to the influence of El Estacio navigation channel. In general,

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the levels of 4-tOP were two times higher than the values determined in spring and

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varied from 11.4 (MM5) to 93.4 µg kg-1 dw. Contrarily, lower concentrations of NP and

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BPA were found in autumn at levels that varied from 67.3 (MMRF2) to 436 µg kg-1 dw

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and from 3.10 (MM6) to 14.7 µg kg-1 dw, respectively. The same way as in spring,

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maximum concentrations of the three compounds corresponded to MM1, demonstrating

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the pollutant impact exerted by 4-APs and BPA on this sampling point. Measured

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concentrations were higher than the previously mentioned criteria for sediments

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reported by European documents (3.4 µg kg-1 dw for 4-tOP and 180 µg kg-1 dw for NP),

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demonstrating the importance of these EDCs as pollutants in the aquatic ecosystem.

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3.2.3. Comparison with other locations

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In comparison with waters, few works about the fate of these EDCs in marine

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sediments can be found in the literature (SM, Table S2). NP is included in all of them,

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while 4-tOP is considered only in 50% and BPA in less than 10%. Linear 4-APs are not

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investigated in any of the reported works and therefore are not taken into account in the

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following discussion.

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Branched 4-APs levels found in our study are in agreement with others carried

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out on the Spanish coast, such as Catalonia (Sánchez-Avila et al., 2011) and other areas 17

ACCEPTED MANUSCRIPT of the Mediterranean Sea (Petrovic et al., 2002). The latter only considered NP, being

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the highest concentration (>1000 µg kg-1 dw) measured near an untreated urban

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WWTP. The occurrence of branched 4-APs was also investigated in different European

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estuaries from UK (Blackburn et al., 1999; Lye et al., 1999), Germany (Bester et al.,

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2001), the Netherlands (Jonkers et al., 2005) and Poland (Ruczyńska et al., 2016). In

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general, higher concentrations were measured in these studies because of the proximity

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of the sampling points to WWTP. The case of the North Sea (Germany) is an exception:

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lower levels of NP were found, probably because the sediments were collected in

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marinas or in the surrounding of marinas. Contamination by 4-APs was also found in

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different areas around the world, at concentrations from two to ten times higher than

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those measured in our study, as in the case of Japan (Ferguson et al., 2001), Korea (Koh

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et al., 2006) and China (Duan et al., 2014; Xu et al., 2018). Concerning BPA, similar

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concentrations were found in China (Diao et al., 2017) whereas higher concentrations

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were measured in Korea, related to industrial discharges from plastic manufacture (Koh

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et al., 2006).

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3.3. Water-sediment partitioning

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The carbon normalised sediment-water partition coefficient (Koc) was used for

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evaluating the capability of branched 4-APs and BPA to be absorbed by sediment from

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seawater (Isobe et al. 2001; Pojana et al. 2007). It can be calculated according to the

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following equation:

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Koc = (Csed/Cwater)/ƒoc

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where Csed and Cwater are concentrations of a compound in respectively sediments and

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water and ƒoc is the mass fraction of the organic carbon on the particles. In this study log

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Koc values were calculated taking into account the both regions and the two campaigns,

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ACCEPTED MANUSCRIPT and were 4.1-4.8 for 4-tOP (mean= 4.5±0.3), 4.4-5.1 for NP (mean= 4.7±0.3) and 3.5-

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3.9 for BPA (mean= 3.8±0.15). These parameters are in agreement with those reported

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in previous studies (Isobe et al., 2001; Pojana et al., 2007). As expected, the log Koc

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values decreased with increasing polarity (BPA<4-tOP
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2010; Xu et al., 2018).

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3.4. Environmental risk assessment

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Table 1 summarizes the potential risk posed by the three selected EDCs on the

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ecosystems of Ria de Vigo and the Mar Menor lagoon, considering the water

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compartment. Results obtained for 4-tOP and BPA demonstrated negligible risk at

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either average (RQm<0.1) and extreme (RQex<0.1) conditions in both estuaries,

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according to the reported levels of concern. In the case of NP, moderate risk was

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observed under extreme conditions (0.1
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times higher in the Mar Menor lagoon. This considerable risk can be attributed to its

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toxicity to aquatic species (from two to ten times lower than 4-tOP and BPA). Although

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NP concentrations complied with EQS established by the European legislation

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(Directive-2013/39, 2013), a moderate risk associated with this compound should not be

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neglected. The results are in agreement with other RQ estimations for water

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compartment, which demonstrate the risk posed by NP (Diao et al., 2017).

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RQ estimated for the sediment compartment were higher than those calculated in

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waters, particularly in the case of branched 4-APs (Table S3, SM). Moderate risk was

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related to 4-tOP when the worst-case scenario of pollution was considered and high risk

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was evidenced for NP. In both cases, RQ observed in the Mar Menor lagoon were

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higher than in Ria de Vigo. No risk was observed for BPA, probably because of its

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lower capability to be accumulated in sediments. Anyway, these results provided

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additional information about the environmental impact posed by EDCs and therefore,

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sediments should be also considered in risk assessment estimations, when possible. Finally, it is worth mentioning that additive or synergistic effects could be

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expected due to the similar disruption mechanisms of target EDCs, being the real hazard

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higher than calculated. Thus, further research is needed to protect the environment and

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to avoid loss of biodiversity in these coastal areas.

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4. CONCLUSIONS

This study provides new data on the occurrence, spatial distribution and seasonal

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variations of 4-APs and BPA in seawaters and sediments from two important and

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impacted Iberian coastal areas, Ria de Vigo (NW Spain) and the Mar Menor lagoon (SE

480

Spain). Linear alkylphenols (4-n-OP and 4-n-NP) were not found in any sampling point,

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whereas branched alkylphenols (4-tOP and NP) and bisphenol A were detected at levels

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of concern in the studied areas.

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Low and moderate concentrations of target compounds (<0.1 µg L−1) were found

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in seawaters, being higher in the Mar Menor lagoon than in Ria de Vigo, likely due to

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their higher water residence time and lower dilution capacity. The EQS established in

486

the Directive 2013/39/EU for NP (2 µg L−1) in water was not exceeded in any case;

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however, the annual average value set for OPs (0.01 µg L−1) was surpassed in almost all

488

sampling points. Further research is needed to obtain more data for 4-tOP in these areas,

489

in order to control chemical pollution. WWTP and industrial discharges and nautical,

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fishing and agricultural activities seemed to be the main sources of contamination by

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these compounds in these areas. The highest concentrations of BPA (>0.3 µg L−1) were

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found in some sampling points related to WWTP and industrial discharges.

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ACCEPTED MANUSCRIPT Because of their adsorption capability, 4-tOP, NP and BPA were also found in

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sediment samples. Concentrations of NP were five times higher than those measured in

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the case of 4-tOP; however, more than 50% of the sampling points exceeded the

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tentative limits established by certain European reports (3.4 µg kg-1 dw for 4-tOP and

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180 µg kg-1 dw for NP). The correlations between EDCs and sediment properties were

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confirmed in Ria de Vigo and the Mar Menor lagoon, especially in the case of NP and

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BPA. Moreover, the sediment water partitioning coefficients were estimated belonging

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the highest value to NP.

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Finally, the risk quotients calculated for water and sediment compartments

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evidenced the risk posed by branched 4-APs (moderate risk by 4-tOP in sediments and

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moderate/high risk by NP in both compartments). Results show the impact of branched

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4-APs and BPA in the studied marine ecosystem and therefore, further extensive studies

505

are required to make the decision on controlling these areas.

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ACKNOWLEDGEMENTS

This work has been financially supported by Xunta de Galicia and potentially

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cofinanced by ERDF (Refs. GRC2013-047; ED431C-2017/28) and by the Spanish

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Ministry of Economy and Competitiveness cofinanced by ERDF (CTM2013-48194-C3-

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R; PCIN:2015-187-C03-03). N. Salgueiro acknowledges Xunta de Galicia and Axencia

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Galega de Innonavión (GAIN) for her postdoctoral fellowship (Modalidade A, 2016).

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Authors would like to thank the technical assistance provided by Tania Tato from

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ECIMAT, the technical staff of the Marine Pollution Department at IEO-Vigo, as well

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as the officers and crew of R/V José María Navaz for assistance in sample collection

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and preparation.

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Table 1. Eco toxicological endpoints and risk quotient estimations (RQ) of 4-tert-octylphenol (4-tOP), nonylphenol (NP) and bisphenol A (BPA) in Ria de Vigo and the Mar Menor lagoon, considering the water compartment NOEC (µg L-1)

Ria de Vigo

PNECwater

Mar Menor lagoon

BPA

Paracentrous lividusa,b

Oncrhynchus mykissc

(µg L )

-

10

30 (LOEC)

1

5

500

40

20

200

100

0.5

RQm

RQx

RQm

RQx

0.01

0.04

0.01

0.04

SC

NP

Isochrysis galbanaa

0.06

0.17

M AN U

4-tOP

-1

10

0.002

Environmental risk

RI PT

Compound

0.034

0.09

0.30

Low risk (RQ<0.1)

Moderate risk in the worstcase scenario (0.1
0.002

0.048 (RQ<0.1)

NOEC: no-observed effect concentrations. Values selected for PNEC estimations are in bold PNECwater: predicted no-effect concentrations in saltwaters

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RQm: Risk quotient estimation based on mean measured concentrations

RQx: Risk quotient estimations based on maximum measured concentration

718 719

AC C

717

EP

Ref: a(Tato et al., 2018), b(Arslan et al., 2007), c(Van den Belt et al., 2003)

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FIGURE CAPTIONS

722 723 724

Fig 1. Location of seawater and sediment sampling points along the (a) Ria de Vigo and (b) Mar Menor lagoon, showing the main urban nuclei, WWTP effluents, rivers and surface watercourses.

725 726 727 728

Fig 2. Levels of alkylphenols and bisphenol A (µg L-1) measured in seawater from (A) Ria de Vigo and (B) Mar Menor lagoon during spring and autumn (2015). In the case of the Ria de Vigo surface (S) and bottom (B) samples were collected, except in sampling point RV1.

729 730 731

Fig 3. Concentrations (µg kg-1 dw) of 4-alkylphenols and bisphenol A measured in sediments from (A) Ria de Vigo and (B) Mar Menor lagoon during spring and autumn (2015).

SC

RI PT

721

M AN U

732 733

AC C

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734

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Fig. 1.

AC C

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TE D

M AN U

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RI PT

737

738 739 740 741 28

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Fig. 2.

744

M AN U

SC

RI PT

745

EP

748

AC C

747

TE D

746

29

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Fig. 3.

M AN U

SC

RI PT

751

AC C

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TE D

752

30

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A)

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Fig. 1

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B)

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Fig. 2 A)

4-tOP

4-n-OP

4-n-NP

NP

BPA

0.2 0.18 0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0

EP

0.48

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Concentration (µg/L)

Spring

B)

4-tOP

4-n-OP

4-n-NP

NP

BPA

RI PT

M AN U

0.27

SC

Concentration (µg/L)

0.34

0.2 0.18 0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0

4-tOP

4-n-OP

4-n-NP

NP

BPA

NP

BPA

Autumn Concentration (µg/L)

0.2 0.18 0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0

Autumn

TE D

Concentration (µg/L)

Spring

0.2 0.18 0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0

4-tOP

4-n-OP

4-n-NP

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Fig. 3 4-tOP

4-n-OP

4-n-NP

NP

BPA

400

RI PT

Concentration (µg/kg dw)

SC 4-n-NP

BPA

EP

100 90 80 70 60 50 40 30 20 10 0

4-n-OP

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Concentration (µg/kg dw)

4-tOP

Autumn

Spring

Autumn

300 200 100 0

Spring

Autumn NP

600

Concentration (µg/kg dw)

B)

500

M AN U

Spring

TE D

A)

Concentration (µg/kg dw)

600 100 90 80 70 60 50 40 30 20 10 0

500 400 300 200 100

0

Spring

Autumn

ACCEPTED MANUSCRIPT Highlights 4-alkylphenols and bisphenol A important pollutants in Ria de Vigo and Mar Menor lagoon

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The highest concentration measured in seawater corresponding to bisphenol A

•

Sediment concentrations of nonylphenol five times higher than other compounds

•

Wastewater treatment plant effluents and nautical activities main sources of pollution

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Potential risk posed by nonylphenol in both estuaries was indicated by risk quotient estimations

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