**2. Estrogenic endocrine disrupting compounds in surface waters**

Accordingly to the National Institute of Environmental Health Sciences (USA), endocrine disrupting compounds are natural or man-made compounds that may mimic or interfere with the function of hormones in the body, producing a variety of adverse effects over the repro‐ ductive, the neurological and the immune systems of humans and both domestic and wild animals [7]. In the case of EDCs with oestrogenic activity, these substances can act: (*i*) on the hypothalamus, inhibiting the release of gonadotropin releasing hormones [8]; (*ii*) on the pituitary, inhibiting the release of gonadotropins [8]; (*iii*) on the gonads, interfering with the production of steroid hormones, namely E2 [9]; (*iv*) on the circulation of endogenous hormones, as these compounds have the ability to bind to the same plasmatic carriers [8]; (*v*) on the same cellular receptors used by the endogenous hormones causing important structural changes [10]. In males of various fish species, situations of *ovotestis*, *i.e*., presence of oocytes in testes, were reported in polluted systems that include some in Portugal [11].

Here, it is exposed the environmental concentrations of sixteen oestrogenic EDCs, which were chosen taking in account the following features: (*i*) *in vivo* and *in vitro* potency, such as the natural oestrogens and EE2 [12]; (*ii*) abundance in terms of incidence and concentration), such as bisphenol A and the alkylphenols (and their ethoxylates) [13, 14], and (*iii*) the ubiquitous, but paradoxically less studied, phytoestrogens [14, 15].

#### **2.1. Oestrogens**

In line with the referred intentions, at the beginning of the millennium the EU adopted legislation — the EU "Water Framework Directive" (WFD) [3] — that was considered very innovative at the time. The WFD called for a comprehensive and integrated approach of water protection and management, having as its ultimate goal that all European waters (continental surface waters, transitional waters, coastal waters and groundwaters) reached good chemical and ecological status within a 15 years period, since the date of publication of that directive. To achieve these requirements, temporal incremental goals were established amongst all EU states to ensure the success of this program. Thus, it was decided *inter alia* to: (*i*) apply all necessary measures to avoid the damage of superficial water bodies; (*ii*) impose fines on violators responsible for deteriorating the status of surface water bodies; (*iii*) achieve good ecological and chemical status for all artificial or heavily modified water bodies; (*iv*) progres‐ sively reduce the pollution of priority compounds, some of these focused in this Chapter, by

However, as the water situation in each EU state was different and unique, since 2000 and up to the present, it has been necessary to make adjustments to overcome this aspect. Indeed, just one year after the WFD 2000/60/EC publication, this directive was updated [4]. In the renovated document (2455/2001/EC), among other measures, it was published a list containing thirtythree harmful compounds which presence in surface waters should be limited or at least/ reduced under limit values [4]. Among these compounds stands out the persistent organic pollutants (POPs), such as, pesticides, polycyclic aromatic hydrocarbons and alkylphenols. Later, the number of harmful compounds were expanded up to forty five and additional environmental quality standards were included in (2008/105/EC) [5]. Nonetheless, because further toxicity details each pollutant are becoming known, the number of compounds in the WFD list tends to increase. In this vein, the current directive 2013/39/EU integrates new substances in its watch lists [6]. Among those are EDCs such as the extremely potent 17β-

limiting their emissions, their discharge and/or runoff into the environment.

154 Toxicology Studies - Cells, Drugs and Environment

oestradiol (E2) and 17α-ethinylestradiol (EE2), as discussed in this Chapter.

were reported in polluted systems that include some in Portugal [11].

**2. Estrogenic endocrine disrupting compounds in surface waters**

Accordingly to the National Institute of Environmental Health Sciences (USA), endocrine disrupting compounds are natural or man-made compounds that may mimic or interfere with the function of hormones in the body, producing a variety of adverse effects over the repro‐ ductive, the neurological and the immune systems of humans and both domestic and wild animals [7]. In the case of EDCs with oestrogenic activity, these substances can act: (*i*) on the hypothalamus, inhibiting the release of gonadotropin releasing hormones [8]; (*ii*) on the pituitary, inhibiting the release of gonadotropins [8]; (*iii*) on the gonads, interfering with the production of steroid hormones, namely E2 [9]; (*iv*) on the circulation of endogenous hormones, as these compounds have the ability to bind to the same plasmatic carriers [8]; (*v*) on the same cellular receptors used by the endogenous hormones causing important structural changes [10]. In males of various fish species, situations of *ovotestis*, *i.e*., presence of oocytes in testes,

#### *2.1.1. Main characteristics and environmental origins*

Both natural and synthetic oestrogens, including oestrone (E1), E2 and EE2 (Figure 1) may induce, even in low (ng/L) concentrations, from mild to extremely harmful effects over the endocrine system [16]. In particular, these compounds have been associated with the occur‐ rence of endocrine disorders such as those over the reproductive system of a wide range of species, including molluscs, crustaceans, fish, birds and mammals [17-22]. Another disruption effect observed in animals, collected from waters containing high levels of oestrogenic contamination, is the decrease of their immune system responses disorders [23]; this phenom‐ enon also seems to occur when humans are exposed to the same type of EDCs [24, 25]. These observations, together with the abovementioned facts, justified the recent incorporation of these compounds in the WFD watching lists [6]. In this sense, and in order to be aware about the concentrations of E1, E2 and EE2 in surface waters, studies were initiated to monitor their presence and appraise their temporal evolution. It is known that the primary sources of these three EDCs are their excretion by urine and faeces [26]. So, these compounds reach the rivers, estuaries and coastlines either through the discharge of effluents coming from sewage treatment plants (STPs) — or directly from sewages (hence untreated) that deliver their content into waterways.

**Figure 1.** Natural and synthetic oestrogens included (E2 and EE2) or to be included (E1) in the watch list of compounds under surveillance by the WFD [6].

Although the concentrations of the referred EDCs are typically in the order of few ng/L (Table 1), it has been experimentally proven that such amounts are potentially harmful [12]. For example, just 4 ng/L of E2 resulted in the formation of *ovotestis* in male Japanese medaka (*Oryzias latipes*) [27]. Also the male mummichog (*Fundulus heteroclitus*) exposed to 100 ng/L of EE2 showed a large increase in the gonadosomatic index, decrease in testosterone production and liver synthesis of vitellogenin [28].

#### *2.1.2. Oestrogens in surface waters*

The highest concentrations of E1 in surface waters were reported in Japan (Tama River) [29], Taiwan (Shui-Dan River) [30] and, China (Pearl Rivers) [31, 32] where its levels ranged from78.7 ng/L to 85.6 ng/L. The highest amounts of E2 were measured in Italy (Venice Lagoon), Japan (Tama River) and Taiwan (Shui-Dan River) where its levels ranged from12 ng/L to175 ng/L. Finally the highest levels of EE2 were found in Spain (Ebre River) [33], China (Pearl Rivers) [31, 32] and Italy (Venice Lagoon) [34] where its levels ranged from 34 to 130 ng/L. From the *in vivo* and *in vitro* experiments referred in section 3.1.1., among many other, it is concluded that those levels are environmental conditions for the occurrence of endocrine disrupting events. In north/central Europe, the surface water concentrations of these com‐ pounds are typically lower than those described in Asia and Brazil (Table 1). Even so, signif‐ icant biological impacts associated with pollution were detected in Europe. For example, roach (*Rutilus rutilus*) and gudgeon (*Gobio gobio*) males caught in rivers and estuaries of the United Kingdom showed high occurrence of *ovotestis* [35-38]. In Portugal, in the early 2000s, *ovotes‐ tis* was observed in flounder (*Platichthys flesus*) and mullets (*Mugil cephalus*) caught from Douro estuary [11]. However, as it can be seen from Table 1, at that time no information existed about the degree of oestrogenic contamination in Iberian western Atlantic coast surface waters - this led to our integrated studies shown in this Chapter, which first data were published in 2009 (Table 2).


Although the concentrations of the referred EDCs are typically in the order of few ng/L (Table 1), it has been experimentally proven that such amounts are potentially harmful [12]. For example, just 4 ng/L of E2 resulted in the formation of *ovotestis* in male Japanese medaka (*Oryzias latipes*) [27]. Also the male mummichog (*Fundulus heteroclitus*) exposed to 100 ng/L of EE2 showed a large increase in the gonadosomatic index, decrease in testosterone production

The highest concentrations of E1 in surface waters were reported in Japan (Tama River) [29], Taiwan (Shui-Dan River) [30] and, China (Pearl Rivers) [31, 32] where its levels ranged from78.7 ng/L to 85.6 ng/L. The highest amounts of E2 were measured in Italy (Venice Lagoon), Japan (Tama River) and Taiwan (Shui-Dan River) where its levels ranged from12 ng/L to175 ng/L. Finally the highest levels of EE2 were found in Spain (Ebre River) [33], China (Pearl Rivers) [31, 32] and Italy (Venice Lagoon) [34] where its levels ranged from 34 to 130 ng/L. From the *in vivo* and *in vitro* experiments referred in section 3.1.1., among many other, it is concluded that those levels are environmental conditions for the occurrence of endocrine disrupting events. In north/central Europe, the surface water concentrations of these com‐ pounds are typically lower than those described in Asia and Brazil (Table 1). Even so, signif‐ icant biological impacts associated with pollution were detected in Europe. For example, roach (*Rutilus rutilus*) and gudgeon (*Gobio gobio*) males caught in rivers and estuaries of the United Kingdom showed high occurrence of *ovotestis* [35-38]. In Portugal, in the early 2000s, *ovotes‐ tis* was observed in flounder (*Platichthys flesus*) and mullets (*Mugil cephalus*) caught from Douro estuary [11]. However, as it can be seen from Table 1, at that time no information existed about the degree of oestrogenic contamination in Iberian western Atlantic coast surface waters - this led to our integrated studies shown in this Chapter, which first data were published in 2009

**EDC Sampling area Concentration (ng/L) References**

Estuarine and coastal waters, The Netherlands 0.1 - 3.4 [26] Scheldt Estuary, Belgium – The Netherlands ND - 10.0 [39] The Netherlands (surface water) <0.30 - 7.2 [40] Rivers in Germany 0.10 - 4.1 [41] Coastal area of Baltic Sea, Germany 0.08 - 0.54 [42] Seine River, France 1.0 - 3.2 [43] Tiber River, Italy 5.0 - 12 [44] Venice Lagoon, Italy <1.20 - 10 [34] Ebro River, Spain ND - 4.9 [56] Llobregat River, Spain <LOD - 22 [45] Llobregat River, Spain 0.82 - 5.81 [46] Thermaikos Gulf, Nothern Aegean Sea, Greece <LOD [47] Buyukcekme watershed, Istambul, Turkey 1.40 - 5.74 [48]

and liver synthesis of vitellogenin [28].

*2.1.2. Oestrogens in surface waters*

156 Toxicology Studies - Cells, Drugs and Environment

(Table 2).

**E1**



NA: Not Available. ND: Not Detected. LOD: Limit of Detection.

**Table 1.** Concentrations of oestrogens in surface waters (minimum-maximum) measured in surface waters worldwide.

#### *2.1.3. Oestrogens in surface waters from the west Iberian coast (Portugal)*

Being Portugal one of the EU members that signed the commitment with the European Commission (EC) to accomplish the directives referred in the WFD document, systematic efforts have been made by our research group to develop and validate methods adequate to the measurement of EDCs in complex environmental matrices (seawater, estuarine and river waters). Also, a complementary effort has been dedicated to both gather all monitoring data and address their potential risk. In this sense, the evaluations done in Portuguese surface waters warned about risks of environmental impacts of oestrogens and can assist the compe‐ tent authorities to take measures to prevent and clean up these habitats from having EDCs, either by eliminating or at least put them at concentrations below those know to be able to promote biological adverse effects.

The results revealed that the average amounts of oestrogens in Portuguese superficial waters were ≈ 6 ng/L for E1 and ≈ 10 ng/L for E2 and EE2. These concentrations, accordingly with the *in vivo* studies referred previously (section 3.1.1.) are not only able of causing disruptive effects in aquatic animals, but also even induce negative impacts in human health [25, 57]. The findings become additionally relevant in view of the fact that these habitats are commonly used by residents and/or tourists, both for recreational and fishing purposes.

Analyzing the values of Table 2, and concerning the concentrations of all evaluated oestrogens, it concluded that habitats we studied so far have chemical quality deficiencies. Table 2 also points that each analysed geographical zone had quite diverse minimum-maximal amounts. This fact corresponds to spatial differences among sampling sites, in line with the presence/ absence of STPs effluents and domestic discharges in the sampled areas. The last inference is directly correlated with the data obtained in the latest national *census*, which revealed a high number of houses (over 17,000) without any sort of connection to sewers [58]. Beyond this, there is also an additional factor that is the huge number of tourists that seasonally arrive to several studied zones located in both west and south of the Iberian Peninsula [59, 60]. Due to drastic increases of the number of inhabitants, that may rise up to 50%, mainly in summer the concentration of oestrogens in surface waters raised significantly, causing seasonal damages in local biota. So, it becomes clear that the physicochemical parameters typically used to assess the quality of surface waters (i.e., temperature, pH, dissolved O2, nitrites, nitrates and phos‐ phates) are not sufficient to guarantee the protection of both environmental and human health.

**EDC Sampling area Concentration (ng/L) References** Ebro River, Spain 30 - 130 [33] Ebro River, Spain ND [56] Llobregat River, Spain ND [46] Tiber River, Italy ND - 1.0 [44] Venice Lagoon, Italy <1.0 34 [34] Thermaikos Gulf, Nothern Aegean Sea, Greece ND [47] Buyukcekme watershed, Istambul, Turkey 11.7 - 14.0 [48] Acushnet Estuary, USA 3.01 - 4.67 [49] South Florida, USA NA [50] Brazilian surface water ND - 25 [51] Tama River, Japan < 0.20 [29] Dan-Shui River, Taiwan 7.53 - 27.4 [30] Yellow River, China NA [52] Pearl rivers, South China ND - 53.4 [31, 32] Songhua River, Northern China ND [54] Yangtze River estuary, China ND - 0.11 [53]

**Table 1.** Concentrations of oestrogens in surface waters (minimum-maximum) measured in surface waters worldwide.

Being Portugal one of the EU members that signed the commitment with the European Commission (EC) to accomplish the directives referred in the WFD document, systematic efforts have been made by our research group to develop and validate methods adequate to the measurement of EDCs in complex environmental matrices (seawater, estuarine and river waters). Also, a complementary effort has been dedicated to both gather all monitoring data and address their potential risk. In this sense, the evaluations done in Portuguese surface waters warned about risks of environmental impacts of oestrogens and can assist the compe‐ tent authorities to take measures to prevent and clean up these habitats from having EDCs, either by eliminating or at least put them at concentrations below those know to be able to

The results revealed that the average amounts of oestrogens in Portuguese superficial waters were ≈ 6 ng/L for E1 and ≈ 10 ng/L for E2 and EE2. These concentrations, accordingly with the *in vivo* studies referred previously (section 3.1.1.) are not only able of causing disruptive effects in aquatic animals, but also even induce negative impacts in human health [25, 57]. The findings become additionally relevant in view of the fact that these habitats are commonly

Analyzing the values of Table 2, and concerning the concentrations of all evaluated oestrogens, it concluded that habitats we studied so far have chemical quality deficiencies. Table 2 also

used by residents and/or tourists, both for recreational and fishing purposes.

NA: Not Available. ND: Not Detected. LOD: Limit of Detection.

158 Toxicology Studies - Cells, Drugs and Environment

promote biological adverse effects.

*2.1.3. Oestrogens in surface waters from the west Iberian coast (Portugal)*

Comparing the values compiled in Tables 1 and 2 it is concluded that, in general, the levels of oestrogens tend to be higher in the west Iberian Peninsula than in the rest of central/north Europe. In fact, in average, these substances in the current study area are almost similar to those measured in many Asian countries. Despite this, it is exalted the positive efforts of remediation conducted in some Portuguese environments, such as those produced in the Ave River, considered in the past as one of the most polluted of Europe [61]. There are also commendable the important efforts occurred in the Douro River estuary [62]. In fact, since our first monitoring studies in the last estuary it was possible to observe that the surface waters collected in 2005 [63] contained significantly higher amounts of oestrogens than those collected in 2009, a fact that demonstrates an important improvement of water quality [62]. Unfortu‐ nately, because there are no other data prior to 2005, with regard to the levels of E1, E2 and EE2 in other Portuguese aquatic systems it is not possible yet to establish with certainty, trend lines relating to a progressive decrease in the concentrations of these EDCs in Portuguese surface waters. In spite of this, other important data provided by our studies demonstrate that: (*i*) several areas commonly seen as "pristine" (e.g., the Mira River) contain high levels of those oestrogens (*unpublished data*); (*ii*) there is an underestimation of the efficacy of the STPs, which at times are not adequately dimensioned, namely for coping with seasonal/touristic influxes; (*iii*) currents from both Atlantic Ocean and/or estuaries can channel pollutants, such as these oestrogens, towards protected areas (e.g., the natural reserve of the Sado River estuary) [59]. From Table 1 it is observed that also in Spain, particularly in the Ebro River for EE2 [33, 56], there are efforts that seem to have been effective in reducing the environmental amounts of these EDCs.

The data repertoire summarized in Table 2 for E1, E2, and EE2 is one of more systematic ones available in the international literature about the oestrogenic status of a particular European country. This information, which can be viewed as a benchmark for the concentrations of oestrogens in Portuguese waters, makes it possible to everybody to monitor the effectiveness of the implementation of measures that may lead to the reduction of environmental levels of those EDCs along the time.


**Table 2.** Concentrations (minimum–maximum) of natural and pharmaceutical oestrogens in Portuguese surface waters.

#### **2.2. Industrial and household products**

**EDC Sampling area Concentration (ng/L) References**

Lima River estuary and Atlantic coast of Viana-Castelo 4.6 - 36.3 [64] Ave River estuary and Atlantic coast of Vila-Conde 0.5 - 7.2 [61] Leça River estuary and Atlantic coast of Porto 4.9 - 10.4 [65] Douro River estuary <15 - 113 [63] Douro River estuary and Atlantic coast of Porto 1.5 - 4.6 [62] Mondego River estuary <5.0 [66] Mondego River and its estuary 1.0 - 14.6 [67] Tagus River and its estuary ≈ 2 - ≈ 6 *Unpublished* Sado River and its estuary 1.0 - 9.8 [59] Mira River and its estuary ≈ 3 - ≈ 12 *Unpublished* Ria Formosa 1.0 - 2.0 [60]

Lima River estuary and Atlantic coast of Viana-Castelo 2.4 - 24.4 [64] Ave River estuary and Atlantic coast of Vila-Conde 1.6 - 9.4 [61] Leça River estuary and Atlantic coast of Porto 3.3 - 5.9 [65] Douro River estuary <7.0 [63] Douro River estuary and Atlantic coast of Porto 5.4 - 8.5 [62] Mondego River estuary <3.0 [66] Mondego River and its estuary 1.5 - 18.4 [67] Tagus River and its estuary ≈ 3 - ≈ 20 *Unpublished* Sado River and its estuary 1.2 - 10.8 [59] Mira River and its estuary ≈ 4 - ≈ 62 *Unpublished* Ria Formosa 1.3 - 10.1 [60]

Lima River estuary and Atlantic coast of Viana-Castelo 0.3 - 19.4 [64] Ave River estuary and Atlantic coast of Vila-Conde 0.3 - 20.4 [61] Leça River estuary and Atlantic coast of Porto 2.1 - 4.4 [65] Douro River estuary 18 - 102 [63] Douro River estuary and Atlantic coast of Porto <1.3 - 4.5 [62] Mondego River estuary <12 [66] Mondego River and its estuary 0.3 - 11.3 [67] Tagus River and its estuary ≈ 4 - ≈ 20 *Unpublished* Sado River and its estuary 1.1 - 3.2 [59] Mira River and its estuary ≈ 4 - ≈ 67 *Unpublished* Ria Formosa 12.1 - 25.0 [60]

**Table 2.** Concentrations (minimum–maximum) of natural and pharmaceutical oestrogens in Portuguese surface

**E1**

160 Toxicology Studies - Cells, Drugs and Environment

**E2**

**EE2**

waters.

#### *2.2.1. Main characteristics and environmental origins*

There are industrial and household compounds prone to promote oestrogenic effects in wildlife and humans [68-70]. Some of these EDCs are compounds such as phenols (bisphenol A, BPA) and alkylphenols (APs) — *viz*. octylphenols (OPs) and nonylphenols (NPs) — and their ethoxylates (APEOs) (Figure 2).


 **Figure 2.** Industrial and household products included in the list of compounds under surveillance by the WFD (Euro‐ pean 2013).

Although the disruptive activity of these compounds is much lower than that of natural and synthetic oestrogens, as they may reach levels in the order of tens to hundreds of µg/L they can became harmful for aquatic fauna. Because of this, and despite great controversy between diverse agents, these compounds become subjected to strict laws that included both APs and APEOs, in the group of "priority substances in the field of water policy 2455/2001/EC" [4]. Presently, the WFD established that the concentration of NPs in surface waters should not exceed 2 µg/L [6].

10

BPA origin in the environment comes from the fact that it is the monomer of the polycarbonate (plastic) used in an huge variety of domestic stuffs and as an intermediate in the synthesis of epoxy resins, flame retardants and many other products [71]. In spite of its vast usage, controversial opinions do exist among those calling for its banning and those that devaluate the BPA toxic effects in realistic scenarios. The February 2007 study "Toxic Baby Bottle's", by the Environment California Research and Policy Center [72], showed that even small amounts of BPA may be one cause of diseases, including breast cancer, prostatic hyperplasia, diabetes, obesity, and hyperactivity disorders involving the immune system. Infertility and early puberty are also among the possible effects caused by BPA, and all of them are associated with the compound's ability to deregulate the endocrine system. "priority substances in the field of water policy 2455/2001/EC" [4]. Presently, the WFD established that the concentration of NPs in surface waters should not exceed 2g/L [6]. BPA origin in the environment comes from the fact that it is the monomer of the polycarbonate (plastic) used in an huge variety of domestic stuffs and as an intermediate in the synthesis of epoxy resins, flame retardants and many other products [71]. In spite of its vast usage, controversial opinions do exist among those calling for its banning and those that devaluate the BPA toxic effects in realistic scenarios. The February 2007 study "Toxic Baby Bottle's", by the Environment California Research and Policy Center [72], showed that even small amounts of BPA may be one cause of diseases, including breast cancer, prostatic hyperplasia, diabetes, obesity, and hyperactivity disorders involving the immune system. Infertility and early puberty are also among the possible effects caused by BPA, "priority substances in the field of water policy 2455/2001/EC" [4]. Presently, the WFD established that the concentration of NPs in surface waters should not exceed 2g/L [6]. BPA origin in the environment comes from the fact that it is the monomer of the polycarbonate (plastic) used in an huge variety of domestic stuffs and as an intermediate in the synthesis of epoxy resins, flame retardants and many other products [71]. In spite of its vast usage, controversial opinions do exist among those calling for its banning and those that devaluate the BPA toxic effects in realistic scenarios. The February 2007 study "Toxic Baby Bottle's", by the Environment California Research

The APEOs sources in the environment are due to their commonly usage as non-ionic surfactant compounds and dispersants [14, 73]. Due to these properties APEOs are being used in detergents and additives of pesticide formulations. Currently, these applications are prohibited in the EU [4] as these compounds are known to readily degrade, in both aerobic and anaerobic conditions, into APs that are more toxic compounds than APEOs (Figure 3). In spite of the APs toxicity, it is known that their lifetime is not long, since they usually degrade in about 10 to 15 hours after sunlight exposure [74]. Thus, it is concluded that the ubiquitous presence of APs in surface waters implies the continuous entrance of APEOs in the environ‐ ment [60]. and all of them are associated with the compound's ability to deregulate the endocrine system. The APEOs sources in the environment are due to their commonly usage as non-ionic surfactant compounds and dispersants [14, 73]. Due to these properties APEOs are being used in detergents and additives of pesticide formulations. Currently, these applications are prohibited in the EU [4] as these compounds are known to readily degrade, in both aerobic and anaerobic conditions, into APs that are more toxic compounds than APEOs (Figure 3). In spite of the APs toxicity, it is known that their lifetime is not long, since they usually degrade in about 10 to 15 hours after sunlight exposure [74]. Thus, it is concluded that the ubiquitous presence of APs in surface waters implies the continuous entrance of APEOs in the environment [60]. and Policy Center [72], showed that even small amounts of BPA may be one cause of diseases, including breast cancer, prostatic hyperplasia, diabetes, obesity, and hyperactivity disorders involving the immune system. Infertility and early puberty are also among the possible effects caused by BPA, and all of them are associated with the compound's ability to deregulate the endocrine system. The APEOs sources in the environment are due to their commonly usage as non-ionic surfactant compounds and dispersants [14, 73]. Due to these properties APEOs are being used in detergents and additives of pesticide formulations. Currently, these applications are prohibited in the EU [4] as these compounds are known to readily degrade, in both aerobic and anaerobic conditions, into APs that are more toxic compounds than APEOs (Figure 3). In spite of the APs toxicity, it is known that their lifetime is not long, since they usually degrade in about 10 to 15 hours after sunlight exposure [74].

Figure 3. Summarized degradation mechanism of APEOs in the environment adapted from Warhurst

Thus, it is concluded that the ubiquitous presence of APs in surface waters implies the continuous

effects were proved, for example, after *in vivo* exposures of male rainbow trout (*Oncorhynchus mykiss*) to 30 g/L of 4-OP, and of male Japanese medaka (*O. latipes*) to 30 g/L of 4-NP, eliciting, respectively, an inhibition of the testicular growth and hermaphroditism [27, 77, 78]. Alas, as it is possible to purchase in EU countries imported products containing APEOs — see an interesting

The endocrine disrupting activity of APEOs and (especially) that of APs, whether they are octylphenols (4-OP and 4-t-OP) or nonylphenols (4-NP and 4-n-NP) is derived from the existence of **Figure 3.** Summarized degradation mechanism of APEOs in the environment adapted from Warhurst [75]. **Alkylphenol Ethoxylate Alkylphenol Monoethoxylate** 

similarities between the structure of these compounds and that of E2 (Figure 4) [76]. Figure 4. Comparison between the chemical structures of E2 (**a**) and 4-NP (**b**). **(a) (b)** The endocrine disrupting activity of APEOs and (especially) that of APs, whether they are octylphenols (4-OP and 4-t-OP) or nonylphenols (4-NP and 4-n-NP) is derived from the existence of similarities between the structure of these compounds and that of E2 (Figure 4) [76]. (APnEO *n*= 0-20) (AP1EO) The endocrine disrupting activity of APEOs and (especially) that of APs, whether they are octylphenols (4-OP and 4-t-OP) or nonylphenols (4-NP and 4-n-NP) is derived from the existence of

similarities between the structure of these compounds and that of E2 (Figure 4) [76].

9

**Alkylphenol**  (AP)

9

possible to purchase in EU countries imported products containing APEOs — see an interesting As such, APs may induce oestrogenic effects using the same paths described in Section 3. These **Figure 4.** Comparison between the chemical structures of E2 (**a**) and 4-NP (**b**).

As such, APs may induce oestrogenic effects using the same paths described in Section 3. These effects were proved, for example, after *in vivo* exposures of male rainbow trout (*Oncorhynchus mykiss*) to 30 µg/L of 4-OP, and of male Japanese medaka (*O. latipes*) to 30 µg/L of 4-NP, eliciting, respectively, an inhibition of the testicular growth and hermaphroditism [27, 77, 78]. Alas, as it is possible to purchase in EU countries imported products containing APEOs — see an interesting record in "APEOs Investigation Report" [79] — these EDCs continue to exist in European surface waters.

Currently, we theorize that the most likely source of APEOs in the aquatic systems is the discharge of wastewater effluents from STPs, nevertheless not neglecting the possible leaching caused by the runoff of these EDCs from landfills and agricultural areas (as APEOs are emulsifiers in some pesticide formulations).

#### *2.2.2. BPA, APEOs and APs in surface waters*

BPA origin in the environment comes from the fact that it is the monomer of the polycarbonate (plastic) used in an huge variety of domestic stuffs and as an intermediate in the synthesis of epoxy resins, flame retardants and many other products [71]. In spite of its vast usage, controversial opinions do exist among those calling for its banning and those that devaluate the BPA toxic effects in realistic scenarios. The February 2007 study "Toxic Baby Bottle's", by the Environment California Research and Policy Center [72], showed that even small amounts of BPA may be one cause of diseases, including breast cancer, prostatic hyperplasia, diabetes, obesity, and hyperactivity disorders involving the immune system. Infertility and early puberty are also among the possible effects caused by BPA, and all of them are associated with

"priority substances in the field of water policy 2455/2001/EC" [4]. Presently, the WFD established

BPA origin in the environment comes from the fact that it is the monomer of the polycarbonate (plastic) used in an huge variety of domestic stuffs and as an intermediate in the synthesis of epoxy resins, flame retardants and many other products [71]. In spite of its vast usage, controversial opinions do exist among those calling for its banning and those that devaluate the BPA toxic effects in realistic scenarios. The February 2007 study "Toxic Baby Bottle's", by the Environment California Research and Policy Center [72], showed that even small amounts of BPA may be one cause of diseases, including breast cancer, prostatic hyperplasia, diabetes, obesity, and hyperactivity disorders involving the immune system. Infertility and early puberty are also among the possible effects caused by BPA, and all of them are associated with the compound's ability to deregulate the endocrine system.

that the concentration of NPs in surface waters should not exceed 2g/L [6].

"priority substances in the field of water policy 2455/2001/EC" [4]. Presently, the WFD established

BPA origin in the environment comes from the fact that it is the monomer of the polycarbonate (plastic) used in an huge variety of domestic stuffs and as an intermediate in the synthesis of epoxy resins, flame retardants and many other products [71]. In spite of its vast usage, controversial opinions do exist among those calling for its banning and those that devaluate the BPA toxic effects in realistic scenarios. The February 2007 study "Toxic Baby Bottle's", by the Environment California Research and Policy Center [72], showed that even small amounts of BPA may be one cause of diseases, including breast cancer, prostatic hyperplasia, diabetes, obesity, and hyperactivity disorders involving the immune system. Infertility and early puberty are also among the possible effects caused by BPA, and all of them are associated with the compound's ability to deregulate the endocrine system.

The APEOs sources in the environment are due to their commonly usage as non-ionic surfactant compounds and dispersants [14, 73]. Due to these properties APEOs are being used in detergents and additives of pesticide formulations. Currently, these applications are prohibited in the EU [4] as these compounds are known to readily degrade, in both aerobic and anaerobic conditions, into APs that are more toxic compounds than APEOs (Figure 3). In spite of the APs toxicity, it is known that their lifetime is not long, since they usually degrade in about 10 to 15 hours after sunlight exposure [74]. Thus, it is concluded that the ubiquitous presence of APs in surface waters implies the continuous

Figure 3. Summarized degradation mechanism of APEOs in the environment adapted from Warhurst

The endocrine disrupting activity of APEOs and (especially) that of APs, whether they are octylphenols (4-OP and 4-t-OP) or nonylphenols (4-NP and 4-n-NP) is derived from the existence of

As such, APs may induce oestrogenic effects using the same paths described in Section 3. These effects were proved, for example, after *in vivo* exposures of male rainbow trout (*Oncorhynchus mykiss*) to 30 g/L of 4-OP, and of male Japanese medaka (*O. latipes*) to 30 g/L of 4-NP, eliciting, respectively, an inhibition of the testicular growth and hermaphroditism [27, 77, 78]. Alas, as it is possible to purchase in EU countries imported products containing APEOs — see an interesting

The APEOs sources in the environment are due to their commonly usage as non-ionic surfactant compounds and dispersants [14, 73]. Due to these properties APEOs are being used in detergents and additives of pesticide formulations. Currently, these applications are prohibited in the EU [4] as these compounds are known to readily degrade, in both aerobic and anaerobic conditions, into APs that are more toxic compounds than APEOs (Figure 3). In spite of the APs toxicity, it is known that their lifetime is not long, since they usually degrade in about 10 to 15 hours after sunlight exposure [74]. Thus, it is concluded that the ubiquitous presence of APs in surface waters implies the continuous entrance of APEOs in the environ‐

The APEOs sources in the environment are due to their commonly usage as non-ionic surfactant compounds and dispersants [14, 73]. Due to these properties APEOs are being used in detergents and additives of pesticide formulations. Currently, these applications are prohibited in the EU [4] as these compounds are known to readily degrade, in both aerobic and anaerobic conditions, into APs that are more toxic compounds than APEOs (Figure 3). In spite of the APs toxicity, it is known that their lifetime is not long, since they usually degrade in about 10 to 15 hours after sunlight exposure [74]. Thus, it is concluded that the ubiquitous presence of APs in surface waters implies the continuous

Figure 3. Summarized degradation mechanism of APEOs in the environment adapted from Warhurst

The endocrine disrupting activity of APEOs and (especially) that of APs, whether they are octylphenols (4-OP and 4-t-OP) or nonylphenols (4-NP and 4-n-NP) is derived from the existence of

The endocrine disrupting activity of APEOs and (especially) that of APs, whether they are octylphenols (4-OP and 4-t-OP) or nonylphenols (4-NP and 4-n-NP) is derived from the existence of similarities between the structure of these compounds and that of E2 (Figure 4) [76].

As such, APs may induce oestrogenic effects using the same paths described in Section 3. These effects were proved, for example, after *in vivo* exposures of male rainbow trout (*Oncorhynchus mykiss*) to 30 g/L of 4-OP, and of male Japanese medaka (*O. latipes*) to 30 g/L of 4-NP, eliciting, respectively, an inhibition of the testicular growth and hermaphroditism [27, 77, 78]. Alas, as it is possible to purchase in EU countries imported products containing APEOs — see an interesting

similarities between the structure of these compounds and that of E2 (Figure 4) [76].

**Alkylphenol Ethoxylate**  (APnEO *n*= 0-20)

entrance of APEOs in the environment [60].

**Figure 3.** Summarized degradation mechanism of APEOs in the environment adapted from Warhurst [75].

Figure 4. Comparison between the chemical structures of E2 (**a**) and 4-NP (**b**).

**(a) (b)**

**(a) (b)**

**Figure 4.** Comparison between the chemical structures of E2 (**a**) and 4-NP (**b**).

[75].

**Alkylphenol Monoethoxylate**  (AP1EO)

similarities between the structure of these compounds and that of E2 (Figure 4) [76].

Figure 4. Comparison between the chemical structures of E2 (**a**) and 4-NP (**b**).

9

9

**Alkylphenol**  (AP)

> **Alkylphenol**  (AP)

**Alkylphenol Monoethoxylate**  (AP1EO)

the compound's ability to deregulate the endocrine system.

entrance of APEOs in the environment [60].

162 Toxicology Studies - Cells, Drugs and Environment

**Alkylphenol Ethoxylate**  (APnEO *n*= 0-20)

that the concentration of NPs in surface waters should not exceed 2g/L [6].

ment [60].

[75].

Table 3 shows an overview of BPA, APEOs and APs in estuaries and rivers worldwide. In line with the broad use of these EDCs it is observed that, at least up to 2005, their concentrations in surface waters were in the order of µg/L. For instance, ten years ago BPA reached concen‐ trations of 5.0 µg/L in several German rivers [80] and the alkylphenols, 4-OP and 4-NP, levels attained 26 to 37 µg/L in several Spanish rivers and estuaries [81]. In line with these observa‐ tions, it could be hypothesized that if there was not the pressure exerted by the program set out by the WFD, together with the directive 76/769/EEC [82] that established restrictions on the marketing and use of NPs and NPEOs, the current environmental content of these EDCs in European surface waters would probably be worse than that found nowadays. It is impor‐ tant to stress that the last directive advocates the banishment of NP and NPEOs in formulations when their levels are equal or higher than 0.1% by mass. This measure leaded to a decrease in the use of NPEOs or even their deliberate discontinuity in Europe (case of Germany) and, consequently, the number of studies that accompanied the temporal evolution of these compounds began decreasing. This aspect increases the difficulty to assess the actual amounts of APEOs and APs in European surface waters, being assumed that their usage was deprecated; a presumption that is not entirely correct, as we have been witnessing.

As shown in Table 3, the levels of APs in several EU countries continue to show potential toxicity (<0.5-37,300 ng/L). This poses obstacles to the compliance defined by WFD, since it seems impossible to achieve concentrations of ≤100 ng/L for the OPs and, ≤300 ng/L for NPs, in all EU states up to 2015. However, it is possible to observe that recent studies revealed that there is positive effort towards the reduction of the global amounts of both APs and BPA in all European countries. So, presently, and also accordingly to the compilation in Table 3, it is observed that in general the levels of APs and BPA are generally higher in Asia and South America than those measured in the EU.

In opposition, and with the exception of the two works [83, 84], before 2005 there were virtually no data about the levels of these EDCs in the west Iberian Peninsula, inc. Portuguese surface waters. These observations leaded to the creation of regular monitoring programs for these compounds, in order to make a general assessment of the situation in various locations and, when possible, monitor their evolution in time. Such temporal results are central to define if the implementation of WFD policies are being successful in the field, alone or together and correlated with biologic outputs (e.g., biomarkers data). The efforts done in the last decade for evaluating BPA, APEOs and APs pollution in Portuguese surface waters from estuaries, rivers and coastal waters are compiled in Table 4.


the implementation of WFD policies are being successful in the field, alone or together and correlated with biologic outputs (e.g., biomarkers data). The efforts done in the last decade for evaluating BPA, APEOs and APs pollution in Portuguese surface waters from estuaries, rivers

**EDC Sampling area Concentration (ng/L) References**

Gulf of Gdansk, Poland 26.9 - 48.1 [85] Estuaries and rivers, The Netherlands <8.8 - 1,000 [40] German Rivers 0.5 - 14 [86] Elbe River and its tributaries, Germany 3.8 - 92.0 [80] Coastal area of Baltic Sea, Germany ND - 5.7 [42] Baden-Württemberg Rivers, Germany <50 - 272 [87] Glatt River, Switzerland 9.0 - 76 [88] Sussex River, England <5.3 - 10 [89] Ebro River, Spain 10 - 20 [33] Ebro River, Spain ND - 61 [56] Llobregat, Cardener, Anoia, Riera de Rubi River, Spain <90 - 2,970 [81] Venice Lagoon, Italy <1.0 - 145 [34] Thermaikos Gulf, Nothern Aegean Sea, Greece 10.6 - 52.3 [47] New Orleans Waters,USA 0.9 - 158 [90] South Florida, USA 4.8 - 32 [50] Brazilian surface water 25 - 84 [51] Coastal waters of Shenzhen, China 11.2 - 776.6 [91] Dianchi Lake, China 35 - 1,081 [92] Liao River, China 12.3 - 116.5 [52] Songhua River, Northern China 8.24 - 263 [54] Pearl rivers, South China 4.35 - 1,390 [31, 32] Yangtze River estuary, China 0.98 - 43.8 [53] River waters of South Korea and seven Asian countries 3.0 - 100 [93] Tama River, Japan 4.8 - 76.3 [29] Coastal waters, Singapore ND - 2,470 [94]

Gulf of Gdansk, Poland <5.0 - 65.9 [85] Estuaries and rivers, The Netherlands <50 - 6,300 [40] German Rivers 0.8 - 54 [86] Baden-Württemberg River, Germany <20 - 189 [87]

and coastal waters are compiled in Table 4.

164 Toxicology Studies - Cells, Drugs and Environment

**BPA**

**OPs**




**Table 3.** Concentrations (minimum–maximum) of industrial and household products in surface waters worldwide.

#### *2.2.3. BPA, APEOs and APs in west Iberian Peninsula surface waters*

**EDC Sampling area Concentration (ng/L) References** Liao River, China 112 - 900.7 [52] Songhua River, Northern China (4-t-NP) 106 - 344 [54] Songhua River, Northern China (4-n-NP) 0.35 - 3.77 [54] Area of Chongqing, China 100 - 7,300 [100] Pearl River, China 36 - 33,000 [31] Tama River, Japan 51.6 - 147.0 [29] River waters of South Korea and 7 Asian Countries <LOD - 2,097 [93]

> The Netherlands (surface water) <160 - 1,700 [40] Elbe River and its tributaries, Germany 0.6 - 9.6 [80] Seine River, France 55 - 63 [101] Ombrone River, Italy (4-t-OP1EO) <94 [95] Ombrone River, Italy (4-t-OP2EO) 6 - 34 [95] Ebro River, Spain (OP2EO) 1.7 - 8.3 [56] Thermaikos Gulf, Greece (OP1EO) <4 - 9.5 [47] Thermaikos Gulf, Greece (OP2EO) <4 - 11.7 [47] Estuaries and rivers, USA 7 - 400 [102]

> Back River, Chesapeake Bay, USA (OP1EO) <0.2 [97] Back River, Chesapeake Bay, USA (OP2EO) <0.02 [97] The Netherlands (surface water) <180 - 8,700 [40] Elbe River and its tributaries, Germany <0.5 - 124 [80] Seine River, France (NP1EO) 9 - 11 [101] Ombrone River, Italy (4-t-NP1EO) <122 [95] Ombrone River, Italy (4-t-NP2EO) 9 - 35 [95] Ebro River, Spain (NP2EO) 9.4 - 275 [56] Thermaikos Gulf, Greece (NP1EO) 15.2 - 270 [47] Thermaikos Gulf, Greece (NP2EO) 14.6 - 346 [47] Estuaries and rivers, USA 220 - 1,050 [102] Back River, Chesapeake Bay, USA (NP1EO) <0.2 - 67 [97] Back River, Chesapeake Bay, USA (NP2EO) 12 - 57 [97] Buenos Aires, Argentina (NP1EO) 100 - 9,200 [99] Buenos Aires, Argentina (NP2EO) 100 - 5,400 [99] Songhua River, Northeastern China (NP1EO) 8.9 - 385 [54] Songhua River, Northeastern China (NP2EO) 19.6 - 321 [54]

**OPEOs**

166 Toxicology Studies - Cells, Drugs and Environment

**NPEOs**

Recent studies revealed that surface waters taken from Portuguese aquatic environments show average concentrations of 650 ng/L for BPA, 1,000 ng/L for APEOs and 360 ng/L for APs (Table 4). Comparing such data with those reported in other countries (Table 3) it is observed that in Portugal the global amounts of these compounds are still quite high. Nevertheless, it is important to note that the values measured before/during 2005 [63, 66, 83, 84], namely for BPA and APs, are significantly higher than those measured in 2010-2011. Thus, and assuming that these results represent the wider reality of the west Iberian Peninsula, it seems that good efforts are being done to reduce the levels of industrial and household pollution (Table 4). In spite of this, Table 4 shows that APEOs exist in amounts that are still approximately one hundred fold higher than those recommended by the European legislation. These observations may be due to the presence of several textile industries and also large agricultural fields located near the sampling areas (Table 4); it should be noticed that several pesticide formulations use APEOs as dispersants. However, since similar amounts of APEOs were found in the Spanish and Greek waters [68, 69] it is possible that these EDCs are still being used, and therefore constitute a global problem of coastal areas.



**EDC Sampling area Concentration (ng/L) References** Ave River estuary and Atlantic coast of Vila-Conde 0.6 - 8.3 [61] Leça River estuary and Atlantic coast of Porto 27.0 - 68.3 [65] Douro River estuary and Atlantic coast of Porto < 3.5 [62] Mondego River and its estuary 0.7 - 1,279 [67] Tagus River and its estuary ≈ 6 - ≈ 150 *Unpublished* Sado River estuary 2.8 - 27.8 [59] Mira River and its estuary ≈ 6 - ≈ 28 *Unpublished* Ria Formosa 3.5 - 8.5 [60] Portuguese rivers and estuaries 0.1 - 30,000 [84]

> Lima River estuary and Atlantic coast of Viana-Castelo 5.7 - 105.0 [64] Ave River estuary and Atlantic coast of Vila-Conde 1.3 - 25.4 [61] Leça River estuary and Atlantic coast of Porto 7.9 - 60.0 [65] Douro River estuary and Atlantic coast of Porto 5.1 - 30.6 [62] Mondego River and its estuary 30 - 27,502 [67] Tagus River and its estuary ≈ 2 - ≈ 71 *Unpublished* Sado River and its estuary 11.4 - 22.0 [59] Mira River and its estuary ≈ 4 - ≈ 27 *Unpublished* Ria Formosa 4.3 - 40.9 [60]

> Lima River estuary and Atlantic coast of V. Castelo 3.0 - 35.4 [64] Ave River estuary and Atlantic coast of V. Conde 0.3 - 16.8 [61] Leça River estuary and Atlantic coast of Porto (north) 28.8 - 63.5 [65] Douro River estuary and Atlantic coast of Porto 3.3 - 116.0 [62] Mondego River and its estuary 20.8 - 2,770 [67] Tagus River and its estuary ≈ 1 - ≈ 41 *Unpublished* Sado River estuary 2.6 - 27.3 [59] Mira River and its estuary ≈ 2 - ≈ 33 *Unpublished* Ria Formosa 3.4 - 14.6 [60]

> Lima River estuary and Atlantic coast of Viana-Castelo 3.9 - 649.8 [64] Ave River estuary and Atlantic coast of Vila-Conde 43.3 - 154.9 [61] Douro River estuary and Atlantic coast of Porto 88.2 - 170.0 [62] Mondego River and its estuary 80.6 - 1,003 [67] Tagus River and its estuary ≈ 200 - ≈ 1,600 *Unpublished* Sado River estuary 129.2 - 239.9 [59] Mira River and its estuary ≈ 52 - ≈ 289 *Unpublished* Ria Formosa 12.2 - 546.6 [60] Portuguese rivers and estuaries 200 - 30,000 [83] Portuguese rivers and estuaries 0.3 - 25,000 [84]

**4-t-OP**

168 Toxicology Studies - Cells, Drugs and Environment

**4-n-NP**

**4-NP**


**Table 4.** Concentrations (minimum–maximum) of industrial and household products in Portuguese surface waters.

#### **2.3. Naturally occurring compounds present in plants**

#### *2.3.1. Phytoestrogens, characteristics and their environmental origin*

Accordingly to the U.S. Food Standards Agency [104], phytoestrogens are "any compound of vegetable origin, or their(s) metabolite(s) with structural similarities to E2, which results in their ability to mimic or block the action of the endogenous sexual hormone in Man". Phy‐ toestrogens fall into two classes: (*i*) flavonoids; and (*ii*) non-flavonoids. The flavonoids are subdivided into the three subclasses: (*i*) isoflavones (Figure 5); (*ii*) coumestans; and (*iii*) prenylflavonoids. The non-flavonoids include the lignans [104].


**Figure 5.** Phytoestrogens with potential oestrogenic activity.

In this Chapter it is focused the group of isoflavones, which include compounds such as daidzein (DAID), genistein (GEN), biochanin A (BIO-A) and formononetin (FORM), due to their structural resemblance with E2. Although not steroids, isoflavones exhibit high affinity for oestrogen receptors. In fact, both *in vivo* and *in vitro* studies have demonstrated their ability to induce abnormal liver synthesis of vitellogenin in male goldfish (*Carassius auratus*) and in rainbow trout (*O. mykiss*) [106-108], and the triggering of hermaphroditism in fish, such as the Japanese medaka (*O. latipes*) exposed to 1,000 µg/L of GEN [15]. Thus, although these com‐ pounds have not been included yet in the list of substances under the supervision by the WFD, but because their global levels in superficial waters can easily reach levels 1,000 times higher than those of E2, i.e., in the order of µg/L or even mg/L, it is plausible to think that phytoes‐ trogens may induce effects equivalent to those described for E2 in spite of having a much lower potency [109]. Despite this, there are not many studies dedicated to the evaluation of these EDCs in surface waters. Also, little information exists about their origins and persistence. Presently, the main source of phytoestrogens in the aquatic environment is attributed either to the presence of the seagrass *Zostera noltii,* and/or to the leaching from the margins where plants rich in these compounds may exist, e.g., *Typha* spp., *Phragmites communis*, *Juncus acutus*, *Fuirena pubescens*, *Carex riparia* and *Carex hispida*, *Cladium mariscus*, *Callitriche stagnalis* and *Potamogeton* spp., *Trifolium* spp*.* and *Papilionaceae* [110-112].

Other equally important source of phytoestrogens in surface waters is their disposal as a result of industrial processes — especially in food processing industries and paper mills [105]. So, as the water levels of these compounds can quickly mount up to mg/L, it is possible that they exert in the wilderness the disruptive biological effects attributed to them [112-114]. Nonethe‐ less, if the levels of these EDCs do not surpass ng/L and, in the absence of other potentially stressful compounds, it is most likely that phytoestrogens are harmless [115].

#### *2.3.2. Phytoestrogens in surface waters*

**2.3. Naturally occurring compounds present in plants**

170 Toxicology Studies - Cells, Drugs and Environment

*2.3.1. Phytoestrogens, characteristics and their environmental origin*

prenylflavonoids. The non-flavonoids include the lignans [104].

**Figure 5.** Phytoestrogens with potential oestrogenic activity.

Accordingly to the U.S. Food Standards Agency [104], phytoestrogens are "any compound of vegetable origin, or their(s) metabolite(s) with structural similarities to E2, which results in their ability to mimic or block the action of the endogenous sexual hormone in Man". Phy‐ toestrogens fall into two classes: (*i*) flavonoids; and (*ii*) non-flavonoids. The flavonoids are subdivided into the three subclasses: (*i*) isoflavones (Figure 5); (*ii*) coumestans; and (*iii*)

In this Chapter it is focused the group of isoflavones, which include compounds such as daidzein (DAID), genistein (GEN), biochanin A (BIO-A) and formononetin (FORM), due to their structural resemblance with E2. Although not steroids, isoflavones exhibit high affinity for oestrogen receptors. In fact, both *in vivo* and *in vitro* studies have demonstrated their ability to induce abnormal liver synthesis of vitellogenin in male goldfish (*Carassius auratus*) and in rainbow trout (*O. mykiss*) [106-108], and the triggering of hermaphroditism in fish, such as the Japanese medaka (*O. latipes*) exposed to 1,000 µg/L of GEN [15]. Thus, although these com‐ pounds have not been included yet in the list of substances under the supervision by the WFD, but because their global levels in superficial waters can easily reach levels 1,000 times higher than those of E2, i.e., in the order of µg/L or even mg/L, it is plausible to think that phytoes‐ trogens may induce effects equivalent to those described for E2 in spite of having a much lower potency [109]. Despite this, there are not many studies dedicated to the evaluation of these EDCs in surface waters. Also, little information exists about their origins and persistence. Presently, the main source of phytoestrogens in the aquatic environment is attributed either Comparatively to the other EDCs, the evaluation of phytoestrogens in aquatic environments, particularly in surface waters of estuaries or rivers, in less studied than the other EDCs referred in this Chapter. This is an important gap in the current knowledge since one the possible origins of these compounds is the natural aquatic flora, which generally is very developed in areas where the organic load is high and prone for eutrophication [116], which is a banal occurrence worldwide. In Table 5 it is shown a collection of studies done in several continents, and the data reveal that the highest concentrations of DAID (42,900 ng/L) and GEN (143,000 n/L) were found in Asia, in the Japanese Kanzaki River. In contrast, the highest amounts of FORM (157 ng/L) and BIO-A (59.4 ng/L) were measured in Europe, at various locations in Switzerland.



ND: Not Detected. LOD: Limit of Detection.

**Table 5.** Concentrations (minimum–maximum) of phytoestrogens in surface waters worldwide.

#### *2.3.3. Phytoestrogens in Portuguese surface waters*

Concerning the Iberian peninsula west Atlantic coast, it was found that surface waters from the Rivers Douro (ca., 19 µg/L BIO-A), Mondego (ca., 5.5 µg/L of FORM and 12 µg/L DAID)

and Tagus (ca., from 10 µ/L) were those holding the higher amounts of phytoestrogens (Table 6). As in these habitats there were occasions when the concentrations of the isoflavones were more than 1,000 times higher than those measured for oestrogens (mainly in spring and summer), it is supposed that those compounds may contribute significantly to endocrine disrupting phenomena occurring in those ecosystems. So, although the phytoestrogens are much less active than oestrogens (E2) their very high concentrations make them worth studying and relevant in monitoring programs.

**EDC Sampling area Concentration (ng/L) References** Waters from Upper Midwest (USA) 1.4 - 1.6 [115] Straight Lake, USA ND [115] Several Rivers, Brazil 3.96 - 336 [122] Waters from Mullet Creek, Australia ND - 1.0 [122] Macquarie Rivulet River, Australia 1.0 - 8.0 [122] Toolijooa surface dam (water), Australia 1.0 - 20 [122] Yeongsan and Seomjin Rivers, Korea ND - 0.7 [93] Salut, Malaysia ND [93] Khong River, Thailand ND [93] Long Xuyen city, Vietnam 1.5 - 2.4 [93] Siem Reap, Cambodia 4.4 [93] Fenhe, China 3.6 - 5.0 [93] Zhangcun River, China ND - 2,650 [124] Kanzaki River (Japan) LOD - 143,000 [123]

> Tiber River, Italy ND [44] Glatt, Töss, Swiss Midlands, Switzerland ND - 217 [117] Surface waters, Switzerland *Detected* - 21 [118] Drainage waters, Switzerland 44 - 157 [118] Several rivers, Iowa, USA 5.3 - 13.5 [120] Straight Lake, USA ND [115] Lake Vadnais, USA 0.9 - 1.1 [115] Macquarie Rivulet River, Australia ND - 2.0 [122] Waters from Mullet Creek, Australia ND - 1.0 [122] Toolijooa surface dam (water), Australia ND - 35 [122]

> Tiber River, Italy 1.0 - 3.0 [44] Glatt, Töss, Swiss Midlands, Switzerland ND - 59.4 [117] Surface waters, Switzerland *Detected* - 12 [118] Drainage waters, Switzerland 7 - 22 [118] Several rivers, Iowa, USA 1.7 - 5.6 [120] Lake Vadnais and Metro Plant effluent channel, USA ND - 1.1 [115] Straight Lake, USA ND [115] Waters from Mullet Creek, Australia ND - 0.1 [122] Macquarie Rivulet River, Australia ND - 1.0 [122] Toolijooa surface dam (water), Australia ND - 4.0 [122]

Concerning the Iberian peninsula west Atlantic coast, it was found that surface waters from the Rivers Douro (ca., 19 µg/L BIO-A), Mondego (ca., 5.5 µg/L of FORM and 12 µg/L DAID)

**Table 5.** Concentrations (minimum–maximum) of phytoestrogens in surface waters worldwide.

**FORM**

**BIO-A**

ND: Not Detected. LOD: Limit of Detection.

172 Toxicology Studies - Cells, Drugs and Environment

*2.3.3. Phytoestrogens in Portuguese surface waters*



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