Anthropogenic contamination of Portuguese coastal waters during the bathing season: Assessment using caﬀeine as a chemical marker Paula Paíga, Cristina Delerue-Matos ⁎ REQUIMTE/LAQV, Instituto Superior de Engenharia do Porto, Politécnico do Porto, Rua Dr. António Bernardino de Almeida, 431, 4200-072 Porto, Portugal ARTICLE INFO Keywords: Atlantic Ocean Bathing season Caﬀeine Human pollution tracer Solid phase extraction UHPLC-ESI-MS/MS ABSTRACT Bathing water quality standards are based on international standards and legislations. However, in Europe, only the microbiological parameters are to be accomplished. Recent research has focused on chemical indicators that can determine human fecal contaminants in water. Therefore, the suitability of caﬀeine as a chemical marker of seawater pollution in the north Portuguese coastal area in the Atlantic Ocean during the bathing season was assessed in this study. The quality of water from the coasts of ﬁve cities was monitored. 101 seawater samples were collected from 14 beaches, and their water quality was classiﬁed as suﬃcient, good, and excellent. Caﬀeine was detected in all samples in concentration range of 18 to 525 ng/L. The highest average concentration of caﬀeine was found in seawater samples collected in July from beaches classiﬁed as having suﬃcient water quality, which were located in cities with high population density and high tourist aﬄuence. Oceans and seas hold about 96.5% of the Earth's water. Although oceans cover two-thirds of the Earth's surface, they are easily vulnerable to human inﬂuences, e.g., overﬁshing, pollution from run-oﬀ, and dumping of waste from human activities (Boopathy, 2000). Sewage eﬄuent from waste water treatment plants (WWTPs) is recognized as a major source of human pollution (Gaw et al., 2013) and is discharged into marine environments through coastal areas and through rivers receiving WWTP eﬄuents (Benotti and Brownawell, 2007). Moreover, because of their location, coastal areas represent a major advantage as many economic activities may take place, including tourism, commer- cial ports, and harbors, thus making them more vulnerable to higher levels of pollution than other seawater samples (Loos et al., 2013). Since the 1970s, the European Union (EU) has laid down rules to safeguard public health and bathing waters. The EU must inform the public about bathing water quality and beach management through bathing water proﬁles. These proﬁles contain information on the kind of pollution and sources that aﬀect the quality of the bathing water and that pose a risk to bathers' health (EC, European Comission, 2015, European bathing water quality). The revised Bathing Water Directive of 2006 updated and simpliﬁed these rules. It requires the Member States to monitor and assess the bathing water quality. The require- ments for award beaches and marine areas with a ﬂag [“Suﬃcient (yellow ﬂag),”“Good (green ﬂag),” and “Excellent” (blue ﬂag)] are: the accessibility, infrastructure, safety of beaches, environmental informa- tion and education, and water quality. The last parameter is a health factor and an important indicator of environmental quality (EC, European Commission, 2006, Directive 2006/7/EC). Concerning the management of bathing water quality, Escherichia coli (fecal coli bacteria) and Enterococcus intestinalis (streptococci) are indicator organisms used for predicting microbiological health risk and to obtain a high level of protection for beaches and marine areas. When “pollution” occurs, it means that there is presence of microbiological contamination or other organisms or waste aﬀecting the quality of the bathing water, presenting a risk to bathers' health (EC, European Commission, 2006, Directive 2006/7/EC). McLella and Eren (2014) stated that the presence of E. coli and E. intestinalis in water is a strong indicator of sewage contamination. However, these analyses do not indicate the origin of the contaminants, i.e., whether it is animal or human origin (Glassmeyer et al., 2005). Suitable markers are therefore necessary to detect and locate the sources of water pollution (Ferreira and da Cunha, 2005). From all the markers investigated such as fecal sterol coprostanol [a fecal sterol produced in the digestive tract of humans by the microbial biohydrogenation of cholesterol (Leeming and Nichols, 1996)] and pharmaceuticals (Daneshvar et al., 2012; Andreu et al., 2016), the one that received the most attention in recent times is caﬀeine (Buerge et al., 2003; Ferreira and da Cunha, 2005; Peeler et al., 2006; Kurissery et al., 2012). Caﬀeine is an ingredient in a variety of beverages (coﬀee, tea, and caﬀeinated soft drinks) and numerous food products (chocolate, pastries, and dairy desserts) (Ferreira, 2005). In the world, 90% of the people consume daily at least one meal or beverage with caﬀeine in it (Buerge et al., 2003). The global average consumption of caﬀeine is