**3. Individual compounds versus total estrogenic load and its endocrine disruption potential**

To better understand and predict the effect of the measured concentrations of all EDCs compiled in this Chapter, in terms of action strength and consequent endocrine disrupting effects, the oestrogenic potency of each compound was estimated relative to that of the standard reference oestrogen, the EE2, the most potent environmental oestrogen at this date. Thus, the average levels of each analysed EDC at every studied area in the west of the Iberian Peninsula were all converted in EE2 equivalents (*EE2eq*). The use of these units facilitates the interpretation of the data. The *EE2eq* estimates of the estrogenic potential of the sixteen EDCs referred in this Chapter followed the next formula [125]:

*E E*2*eq* =*CxF*

Here, *C* concerns to the measured concentration of a given EDC and *F* refers to the EE2 equivalency factor, as determined from *in vitro* assays [125]. Although this type of interpreta‐ tion is very useful, the *C* may vary with the assay and it does not exempt the *in vivo* testing.

Interpreting the results presented here, in light of that normalization, and joining the EDCs by groups (i.e., oestrogens, APs + BPA, APEOs and phytoestrogens) it can be deduced that before 2005 the Portuguese surface waters taken from the rivers Douro, Mondego and Sado exhibited values of *EE2eq* that "hovered" between 24 and 198 ng/L, being the Douro River estuary the habitat with the highest oestrogen load (Table 7). After 2005, possibly due to the application of some of the regulations proposed by WFD, it was observed a significant decrease of the EE2eq in the Douro River estuary surface waters, which displayed values that stand in 12 ng/L, even considering a larger spectrum of analysed EDCs. For the Mondego and Sado Rivers it is also observed that even analysing almost the twice number of EDCs, the data obtained from surface waters in 2005 [66, 103] had similar *EE2eq* (24 ng/L) than those observed in waters from the same sampling areas in 2010-2011. Besides, from the analysis of Table 7 it is also possible to observe which group of compounds contribute the most to the final values of *EE2eq*. Thus, it is concluded that by order of importance the compounds that contribute the most for the *EE2eq* values in the Portuguese surface waters were: (*i*) oestrogens; (*ii*) phytoestrogens; (*iii*) APs + BPA; and (*iv*) APEOs. So, both oestrogens and phytoestrogens are important "key points" to consider when the purpose of achieving good water quality by 2015 is the main goal of the European Environment Agency (EEA). Overall, it is proposed herein that an improvement of the sewerage system could surely promote reduction of the concentration of oestrogens and eutrophication. In addition, it is also suggested that the authorities should equate ways to reduce impacts caused by the use of products containing APEOs, e.g., by regulating their imports.

**EDC Sampling area Concentration (ng/L) References** Mondego River estuary <8.4 - 60 [66] Mondego River and its estuary 50.1 - 590.0 [67]

**Table 6.** Concentrations (minimum–maximum) of phytoestrogens measured in Portuguese surface waters.

**3. Individual compounds versus total estrogenic load and its endocrine**

To better understand and predict the effect of the measured concentrations of all EDCs compiled in this Chapter, in terms of action strength and consequent endocrine disrupting effects, the oestrogenic potency of each compound was estimated relative to that of the standard reference oestrogen, the EE2, the most potent environmental oestrogen at this date. Thus, the average levels of each analysed EDC at every studied area in the west of the Iberian Peninsula were all converted in EE2 equivalents (*EE2eq*). The use of these units facilitates the interpretation of the data. The *EE2eq* estimates of the estrogenic potential of the sixteen EDCs

*E E*2*eq* =*CxF*

Here, *C* concerns to the measured concentration of a given EDC and *F* refers to the EE2 equivalency factor, as determined from *in vitro* assays [125]. Although this type of interpreta‐ tion is very useful, the *C* may vary with the assay and it does not exempt the *in vivo* testing.

Interpreting the results presented here, in light of that normalization, and joining the EDCs by groups (i.e., oestrogens, APs + BPA, APEOs and phytoestrogens) it can be deduced that before 2005 the Portuguese surface waters taken from the rivers Douro, Mondego and Sado exhibited values of *EE2eq* that "hovered" between 24 and 198 ng/L, being the Douro River estuary the habitat with the highest oestrogen load (Table 7). After 2005, possibly due to the application of some of the regulations proposed by WFD, it was observed a significant decrease of the EE2eq in the Douro River estuary surface waters, which displayed values that stand in 12 ng/L, even considering a larger spectrum of analysed EDCs. For the Mondego and Sado Rivers it is also observed that even analysing almost the twice number of EDCs, the data obtained from surface waters in 2005 [66, 103] had similar *EE2eq* (24 ng/L) than those observed in waters from the same sampling areas in 2010-2011. Besides, from the analysis of Table 7 it is also possible to observe which group of compounds contribute the most to the final values of *EE2eq*. Thus, it is concluded that by order of importance the compounds that contribute the most for

Tagus River and its estuary ≈ 6 - ≈ 85 *Unpublished* Sado River and its estuary 10 - 30 [103] Sado River and its estuary 130.8 - 844.5 [59] Mira River and its estuary ≈ 5 - ≈ 460 *Unpublished* Ria Formosa 91.2 - 261.4 [60]

**BIO-A**

**disruption potential**

174 Toxicology Studies - Cells, Drugs and Environment

referred in this Chapter followed the next formula [125]:


Data not available (NA) or (\* ) summations containing different number of analysed EDCs.

**Table 7.** Estimation of the estrogenic potential of several Portuguese surface waters.

## **4. Status of waters in the European Union and in Portugal**

Since the beginning of the implementation of the WFD, the EC was aware that it would not be an easy task to attain the proposed quality goals stated in all EU member states within a limited period of time — 15 years [3]. Therefore, although strict targets ought to be accomplished in all states, it was considered some temporal flexibility to completely achieve the main goals, as it was considered that each nation has its own environmental (and social) characteristics. In this vein, a 2007 report from the EC revealed that nineteen EU states still showed significant weaknesses in the implementation of the WFD and, called attention to the risk of the purposes set for 2015 may not be met. Therefore, in order to coerce the accomplishment of the WFD requirements, the EC appointed the EEA as a periodic gauge which role has been the evaluation of the water quality in each country that assumed to apply the WFD. In this context, during the last evaluation by the EEA, Portugal was identified as having not yet implemented a plan for the management of all national watersheds (Judgment from 21 June 2012 in Case C-223/11, Portugal) [126]. As this task was considered essential for the implementation of various articles defined in the WFD, including the Article 8 that aims the implementation of standards for water monitoring, Portugal together with others (Spain, Greece and Luxembourg) were condemned by the Court of Justice of the European Community. Besides this occurrence, in 2013 the WFD published a list of other, most common, defaults recorded in many states [127]: (*i*) existence of severe gaps in the levels of chemical pollutants from anthropogenic origins in surface waters; (*ii*) 60% of groundwater resources in cities were over-exploitation; (*iii*) 25% of the groundwater was polluted; (*iv*) 47% of the surface waters showed bad ecological status; and (*v*) 50% of the wetlands showed extinction risks of indigenous species. Considering the first item of this list, it is demonstrated the need of implement chemical monitoring programs for all states involved in the implementation of the WFD. As a corollary, we do emphasize herein the relevance of the regular chemical monitoring and the implementation of strategies for reducing the levels of the EDCs referred in this Chapter.
