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Article

Predicting the Ecological Risks of Phytoestrogens in Coastal Waters Using In Silico and In Vitro Approaches

by
Luciana Lopes Guimarães
1,*,
Bárbara Faria Lourenço
1,
Fabio Hermes Pusceddu
2,
Fernando Sanzi Cortez
2,
Rafael Barreiros Kiyotani
1,
Gilmar Aparecido dos Santos
1,
Walber Toma
1 and
Vinicius Roveri
1,3,4
1
Laboratório de Pesquisa em Produtos Naturais, Universidade Santa Cecília (UNISANTA), Rua Oswaldo Cruz 266, C21, Bloco C, Santos 11045-907, SP, Brazil
2
Laboratório de Ecotoxicologia, Universidade Santa Cecília (UNISANTA), Rua Oswaldo Cruz 266, Santos 11045-907, SP, Brazil
3
Universidade Metropolitana de Santos (UNIMES), Avenida Conselheiro Nébias, 536-Encruzilhada, Santos 11045-002, SP, Brazil
4
Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR/CIMAR), Avenida General Norton de Matos S/N, 4450-208 Matosinhos, Portugal
*
Author to whom correspondence should be addressed.
Coasts 2024, 4(4), 651-666; https://doi.org/10.3390/coasts4040034
Submission received: 31 July 2024 / Revised: 21 September 2024 / Accepted: 4 October 2024 / Published: 15 October 2024
Browse Figure
Versions Notes

Abstract

:
Emerging pollutants, like phytoestrogens, are gaining attention in the scientific community for their impact on aquatic organisms. Nevertheless, there is a paucity of studies examining their effects on tropical aquatic species. In this context, the objective of this study was to (i) conduct chronic ecotoxicological assays with the sea urchin Echinometra lucunter with two phytoestrogens, namely genistein and daidzein (both derived from soy plant), and compare the results to the synthetic estrogen ‘estradiol valerate’; (ii) predict the potential risks of these phytoestrogens through an ecological risk assessment; and (iii) create a prioritization list of the most hazardous phytoestrogens using environmental persistence, bioaccumulation, and toxicity (PBT criteria). The results of chronic exposure demonstrated the following order of toxicity: daidzein (IC50 = 2.60 mg/L); genistein (IC50 = 3.37 mg/L); and estradiol valerate (IC50 = 28.40 mg/L). The results classify genistein and daidzein as “toxic” and estradiol valerate as “harmful” to the sea urchin. The final ranking of the PBT approach in coastal waters was as follows: biochanin A (the highest priority), followed by formononetin, genistein, enterolactone, daidzein, estradiol valerate, coumestrol, and 8-prenylnaringenin. The dataset highlights the importance of environmental monitoring to track phytoestrogens in Latin American coastal areas, particularly in developing countries.

1. Introduction

Soybeans (Glycine max) are a rich source of carbohydrates, fats, minerals, and proteins. In recent decades, they have become a dietary staple in numerous countries worldwide [1,2,3]. In addition, soy is increasingly consumed in human diets worldwide due to the adoption of vegan diets [4]. The International Grains Council (IGC) has reported that global soybean consumption during the 2022/23 season reached 359 million tons, with soy being utilized to produce over 400 industrial products [1,2,3].
In this context, genistein (GEN) and daidzein (DAI)—soy isoflavones—are the most extensively researched and well-known compounds present in soybeans [2,3,5]. Undoubtedly, both isoflavones offer significant benefits to human health [1,6]. They are particularly efficacious in the treatment of cancers related to hormonal imbalance, menopausal symptoms, cardiovascular disease, osteoporosis, and hypercholesterolemia [1,7,8].
It is also noteworthy that both isoflavones are classified as phytoestrogens—polyphenolic secondary metabolites that induce significant biological responses in animals [1]. Phytoestrogens have a similar chemical structure to steroid hormones, such as 17β-estradiol. This means they can interact directly with estrogen receptors, affecting the body in ways that are either estrogenic or antiestrogenic [9,10].
The presence of phytoestrogens in plants that serve as food has been the subject of scientific interest since 1940 when infertility in goats in Australia was first observed [11]. Although studies have demonstrated that the consumption of phytoestrogens at low dosages (e.g., 20 mg per day) does not result in any observable reproductive effects in adults, an increase in hypothyroidism manifestations was observed in adults receiving 16 mg per day of the phytoestrogens [12]. Furthermore, recent research has indicated that exposure to phytoestrogens before menopause may result in adverse effects on reproductive physiology [13].
Studies proved that after consumption, phytoestrogens undergo a multitude of metabolic conversions [14]. Metabolites enter the bloodstream and are subsequently excreted in human urine [14,15]. As a result, they are continuously discharged into sewage treatment plants (STP) and aquatic ecosystems worldwide [11,16,17,18,19,20,21]. These chemicals are released at concentrations below the threshold of toxicity, typically in the range of nanograms per liter, from a variety of anthropogenic sources [11,16,17,18,19,20,21]. For instance, GEN and DAI were detected in the Kanzaki River in Japan (maximum detected concentration: 143,400 ng/L of GEN and 42,900 ng/L of DAI) [19]; in the Zhangcun River in China [range = nd (not detected)–2650 ng/L of GEN and nd–1490.0 ng/L of DAI] [21]; and in the Mondego River in Portugal (range = nd–507.1 ng/L of GEN and nd–526 ng/L of DAI) [20].
A review study conducted by Jarošová et al. [11] also corroborated the widespread quantification of GEN and DAI in a multitude of locations across the globe. These include the Douro estuary in Portugal, the Toolijooa Pond in Australia, the Tiber River in Italy, the Vadnais Lake in the USA, the Rhine River in Germany, the Fenhe River in China, the Siem Reap River in Cambodia, the Ton River in Laos, and the Cikamasan River in Indonesia. However, these compounds have significant environmental implications because they are bioactive even at low concentrations [1,22,23]. For instance, studies have shown that freshwater fish species of the genus Oryzias (such as O. latipes) and the genus Betta (such as B. splendens) become more aggressive when exposed to phytoestrogens. This is likely due to a reduction in testosterone and immunosuppression [6,22].
The response of sexually mature Pimephales promelas fish to environmentally relevant concentrations of GEN and DAI was also observed by Rearick et al. [23]. The authors found that larvae exposed to GEN show a notable decline in survival rates, while adult females exposed to DAI demonstrate an increase in egg production. It is also worth noting that the administration of a semi-synthetic diet enriched with 500 mg/L GEN to rainbow trout (Oncorhynchus mykiss) resulted in alterations to gonadotropin hormone levels in fish farm settings [24]. Lu et al. [25] observed a process of feminization in adult male goldfish (Carassius auratus) exposed to water resources from China, with elevated plasma concentrations of 17β-estradiol and the induction of vitellogenin. Similarly, high vitellogenin levels were observed in Salmo trutta in streams from Denmark [25].
Notwithstanding the considerable body of analytical data on the occurrence and the ecotoxicological effects of GEN, DAI, and 17β-estradiol in freshwater ecosystems [11,16,17,18,19,20,21], to the best of our knowledge, there is still a paucity of studies analyzing the potential adverse effects of these compounds on non-target marine aquatic biota. As the global population continues to grow and urbanize, there has been an accompanying increase in the discharge of municipal sewage into marine aquatic ecosystems, especially in coastal areas [26,27,28]. The sewers contain a wide variety of chemicals, including thousands of different substances with highly diverse compositions. Among these compounds are phytoestrogens, which have the potential to degrade the quality of the receiving water bodies [29,30]. The lack of consistent data on the ecotoxicity of GEN, DAI, and 17β-estradiol makes it difficult to assess the potential ecological risks associated with these phytoestrogens. This complicates the development of regulatory guidelines to address coastal aquatic contamination resulting from their presence.
In light of the previous research gaps, the objectives of this study were as follows: (i) conduct chronic ecotoxicological assays utilizing the sea urchin Echinometra lucunter (E. lucunter) as the test organism with two phytoestrogens, namely GEN and DAI (both derived from the soy plant), and compare the results with ecotoxicological assays performed with the corresponding synthetic compound (with a similar therapeutic indication) “estradiol valerate (EV)”. It is important to mention that EV is a synthetic 17β-estradiol that is chemically and biologically identical to natural estradiol; (ii) predict, through an ecological risk assessment, the potential chronic risks of these phytoestrogens, considering the toxicity results with the sea urchin; and (iii) create a prioritization list of the most hazardous phytoestrogens based on the intrinsic properties of these compounds, i.e., environmental persistence in STP (P), bioaccumulation (B), and toxicity (T) (PBT criteria). This dataset serves to reinforce the necessity of implementing continuous monitoring programs for tracking phytoestrogens in coastal zones, particularly in Latin America.

2. Materials and Methods

2.1. Chemical Reagents

In this study, DAI and GEN were selected as the components for investigation through in silico and in vitro approaches, given their established role in the pharmacological activity of soybeans as a medicinal resource (due to their documented action on human estrogen receptors). The EV selection was based on its therapeutic indication for treating menopausal symptoms (also acting on human estrogen receptors), aiming to compare its environmental impact with soybean components (DAI and GEN). All the phytoestrogens were sourced from Sigma-Aldrich (St. Louis, MO, USA). Moreover, to more accurately assess the potential risks associated with GEN, DAI, and EV, five additional globally consumed phytoestrogens were selected for comparison purposes with GEN, DAI, and EV. These included 8-prenylnaringenin, coumestrol, enterolactone, biochanin A, and formononetin. The physicochemical information of these five phytoestrogens was obtained from peer-reviewed literature, and only in silico evaluations were performed for these five phytoestrogens.

2.2. Data on the % Removal of the Phytoestrogens in Sewage as Well as Their Bioaccumulation Potential

This assessment is based on the methodology proposed by Roveri and Guimarães [31], which is outlined below.

2.2.1. Sewage Treatment Plants (STPs): % Removal of the Phytoestrogens

The initial parameter was established using the EPI Suite program (Version 4.11), which was created by the US Environmental Protection Agency (USEPA) [32]. To initiate the in silico predictions, the SMILES string must be provided as input data (Table 1) [33]. To ascertain the behavior (i.e., persistence) of phytoestrogens in a sewage treatment plant, the “STP Total Removal” method was employed [34].

2.2.2. Bioaccumulation Potential of the Phytoestrogens

To assess the bioaccumulation potential of phytoestrogens in soil, sediment, or sludge, the octanol-water partition coefficient (KOW) value was employed [32]. The following guidelines should be adhered to: A log KOW value of less than 2.5 indicates that the phytoestrogen has a low bioaccumulation potential. If the log KOW value falls between 2.5 and 4.5, the chemical is deemed to have moderate bioaccumulation potential. A log KOW of 4.5 or greater indicates that the phytoestrogen is potentially bioaccumulative [35]. Additionally, the EPI Suite was employed to ascertain the bioaccumulation potential of the phytoestrogens [32].

2.3. Chronic Ecotoxicity of Phytoestrogens in the Sea Urchin Echinometra Lucunter

2.3.1. Ecotoxicological Assessment: Obtaining Organisms and the Experiments

The ecotoxicological assessment procedures were conducted following the methodology outlined by Cid et al. [36]. Toxicity assays were performed to evaluate the chronic effects on the embryo–larval development of E. lucunter of two phytoestrogens (GEN and DAI) and EV. The sea urchins were sourced directly from the seawater in Guarujá, São Paulo, Brazil. Following collection, the specimens were stored in a thermal box and transported to the Laboratory of Ecotoxicology at Santa Cecilia University in Santos, Brazil. They were kept in a tank with temperature control and aeration until the time of testing. The water used in the tanks was sourced from Guarujá seawater, which has a natural origin. The physical and chemical parameters of the environment in which the organisms were maintained were observed daily and adjusted as necessary to ensure that the conditions remained within the parameters set out by the Brazilian NBR 15350 standard [37]. To guarantee the complete solubilization and conservation of the attributes inherent to the organisms’ natural habitat, the dilution of test substances, the handling of gametes, and the preparation of control preparations were conducted using reconstituted water from a mixture of commercial salt and processed water (CORAL PRO SALT brand, RED SEA®, São Paulo, Brazil). The water was maintained at physical-chemical standard values established by NBR 15350. The pH level was found to be between 7.8 and 8.4, while the salinity was recorded at a range of 30 to 37 g per liter [37]. To ascertain the impact of the solvent (dimethyl sulfoxide, DMSO) on the results of the phytoestrogen assays, seawater and solvent control groups were set up in parallel.

2.3.2. Chronic Toxicity Tests (Embryo–Larval Development Assay)

Newly fertilized sea urchin embryos were exposed to different nominal concentrations of GEN and DAI, ranging from 1.87 to 30 mg/L, and EV, ranging from 6.25 to 100 mg/L, during the embryo–larval development period, that is, from 36 h to 42 h E. lucunter, following the technical standard ABNT/NBR 15350 [37]. After completing the tests, the larvae were separated into two groups based on their morphological characteristics to distinguish between normal and abnormal larvae [37]. The test reading involved counting the first 100 organisms based on their developmental stages.
The results are expressed as follows: (i) IC50 (medium inhibitory concentration); (ii) NOEC (no observed effect on the concentration of the test organism); and (iii) LOEC (lowest observed concentration that causes a statistically significant effect on the test organisms) [37].
The set of IC50 (42 h) for the embryo–larval development assays was calculated using the linear interpolation method (ICPIN 2.0 program). For each embryo–larval development assay, ANOVA followed by Dunnett’s test was used to identify the concentrations that were significantly different from the control group (NOEC and LOEC). In all the analyses, significant differences were determined when p < 0.05. Statistical analysis was performed using TOXSTAT (version 3.5) [38].
Moreover, the findings revealed no statistically significant difference between the water control and the highest concentration of DMSO solvent (p ≤ 0.05) [37].

2.4. Ecological Risk Assessment of the Phytoestrogens

To ascertain the potential risk of phytoestrogens to aquatic biota, an ecological risk assessment was conducted following the methodology established by Roveri et al. [27,28]. The risk quotient (RQ) was calculated by dividing the maximum measured environmental concentration (MEC) by the predicted no-effect concentration (PNEC) (both expressed in ng/L). The MEC values were sourced from peer-reviewed publications. In this context, the search terms employed were as follows: “occurrence”, “phytoestrogens”, “phenolic flavonoids”, “soy isoflavone”, “genistein”, “daidzein”, “estradiol valerate”, “freshwater”, “coastal waters”, and “seawater”. The search was conducted between the years 2000 and 2023 using a variety of academic search engines. The objective of this search was to identify a database of phytoestrogens that have been detected in coastal ecosystems. In contrast, the PNEC values were obtained using ecotoxicity tests with the sea urchin performed with the phytoestrogens under investigation in the present study. Subsequently, the PNEC values for the chronic toxicity data were calculated by dividing each toxicological endpoint by an assessment factor (AF). The use of an AF is intended to address the inherent uncertainties in extrapolating results from single-species laboratory data to a multi-species ecosystem [39]. By the guidelines set forth by the European Chemicals Agency [40] and the European Chemical Bureau [41], an AF of 100 should be considered in marine environments when evaluating long-term datasets. This implies that the toxicity in question is assumed to be 100 times higher than the values determined in standard tests.
Ultimately, the final risk assessment of phytoestrogens to aquatic biota was classified as insignificant if the RQ was less than 0.01. If the RQ was between 0.01 and 0.1, the risk was classified as low (indicated in green). If the RQ was between 0.1 and 1.0, the risk was classified as moderate (indicated in yellow). If the RQ was greater than 1.0, the risk was classified as high (indicated in red) [42].

2.5. Prioritization Procedure Applied to Phytoestrogens

The prioritization procedure applied to phytoestrogens was adapted from a previous study by Roveri et al. [28]. Each phytoestrogen was attributed to three ranks, from 1 to 5, based on three criteria: persistence, bioaccumulation, and toxicity (PBT). For the persistence and bioaccumulation criteria, the “STP Total Removal” endpoint (described in Section 2.2.1) and the “Log KOW” (described in Section 2.2.2) were employed, respectively. The chronic values (Chv; expressed in mg/L) were calculated using the following equation: Chv = 10[log (NOEC × LOEC/2)]. The ECOSAR program (version 2.0; included in EPI Suite) was employed to ascertain the ChV values of the phytoestrogens [32]. The final ranking for PBT was subsequently determined by the addition of the ranks assigned to the three criteria, as detailed in Table 2.

3. Results and Discussion

3.1. Results of the Chronic Toxicity Tests (Embryo–Larval Development Assay)

This study represents the initial investigation into the adverse biological effects of two phytoestrogens, namely GEN, DAI, and EV (for comparison purposes), on the sea urchin E. lucunter through embryo–larval development assays (chronic toxicity tests).
The data obtained in the present study demonstrate unambiguously that concentrations of GEN and DAI in the order of mg/L significantly inhibit the embryonic and larval development of E. lucunter (see Table 3). The IC50 value for GEN was 3.37 mg/L, with a NOEC of <1.87 mg/L and a LOEC of 1.87 mg/L. The IC50 value for DAI was 2.60 mg/L, with a NOEC of <1.87 mg/L and a LOEC of 1.87 mg/L. Finally, the IC50 value for EV was 28.40 mg/L, with a NOEC of <6.25 mg/L and a LOEC of 6.25 mg/L (Table 3). Following the criteria established by the ‘Technical Guidance Document’ (in Support of Commission Directive 93/67/EEC) set by the Commission of the European Communities [43], the findings of chronic exposure indicate that GEN and DAI may be classified as “toxic” and that EV may be designated as “harmful” to E. lucunter.
It is worth noting that this study revealed that the phytoestrogens DAI and GEN exhibited greater toxicity to E. lucunter compared to EV. This observation is particularly significant considering that estradiol is a well-known endocrine disruptor in aquatic organisms, including sea urchins [44].
Isoflavonoids, such as GEN and DAI, deserve special attention as they are the most well-known of the soy isoflavones [2,3,5]. While most research on phytoestrogens has shown that they have beneficial effects on human health [1,6,7,8], some studies have also shown that most phytoestrogens act as endocrine disruptors, which could potentially result in intersexuality in aquatic organisms [45,46].
In light of the potential adverse effects outlined, considerable attention has been devoted to the occurrence and fate of phytoestrogens in coastal waters, where they have been identified with considerable frequency. For example, they have been identified in the Baltic Sea (GEN: 0.61 ng/L; DAI: 0.43 ng/L) [47] and the Mondego River estuary (GEN: 826.00 ng/L; DAI: 270.0 ng/L) (Portugal) [48]. In 2016, the presence of GEN and DAI was detected in the Douro River estuary (Portugal) (GEN: 135.0 ng/L; DAI: 277.4 ng/L) [10]. Additionally, these phytoestrogens were identified in a coastal lagoon (Portugal) (GEN: 64.3 ng/L; DAI: 147.0 ng/L) [49]. Their presence was also confirmed in the Pearl River estuary (China) (GEN: 12.90 ng/L; DAI: 1.90 ng/L) [50].

3.2. Results of the Ecological Risk Assessment of Phytoestrogens

It is important to assess the potential risks associated with the release of phytoestrogens into coastal aquatic ecosystems to safeguard these crucial environments [36,51]. Following the Technical Guidance Document on Risk Assessment of the European Union [52], an ecological risk assessment screening was conducted for GEN and DAI, considering the worst-case scenario. The MEC values in coastal waters were obtained from peer-reviewed publications. The following sources were consulted: Beck et al. [47], Rocha et al. [48], Ribeiro et al. [10], Rocha et al. [49], and Deich et al. [50] (Table 4).
After reviewing the literature, no studies have been found that detected EV in coastal waters. Consequently, the risk assessment was not performed for this compound.
Regarding the PNEC, these data were calculated based on the results obtained in the present study, specifically from the chronic toxicity tests performed with E. lucunter. The results revealed that 50% of the risk assessments using GEN and DAI displayed low toxicity to E. lucunter, indicating that both deserve attention in coastal waters (Table 4). Specifically, the mentioned toxicity was observed on the Portuguese coast, namely in the estuary of the Mondego River, the estuary of the Douro River, and a coastal lagoon [10,48,49].

3.3. Prioritization of the Phytoestrogens in Coastal Waters

In recent decades, the main source of phytoestrogen contamination in aquatic ecosystems has been identified as outputs from STPs [16,17,18] due to its excretion in urine and feces [14,15]. Approximately 85% of ingested isoflavones are excreted in urine within 48h of ingestion [14,15]. Consequently, phytoestrogens may end up in a conventional activated sludge treatment plant (CASTP; standard system used in many cities worldwide), where they may suffer further biological degradation [32,34]. However, a CASTP is designed to reduce levels of dissolved organic carbon, phosphates, and nitrates but not residues of phytoestrogens [32,34]. For instance, the occurrence of GEN (range = 2.7–38.1 ng/L) and DAI (mean < 10 ng/L) has been confirmed in municipal STP effluent from Germany [17,18]. Additionally, GEN has been detected in wood pulp mill effluent from Canada (with concentrations ranging from 10.5 to 13.1 μg/L) [16]. In this context, the application of the endpoint “STP Total Removal” revealed inefficient removal of GEN and DAI. Specifically, less than 5% of these phytoestrogens were removed in the CASTP. From this percentage, less than 5% of both compounds were adsorbed in the sewage sludge (i.e., only 0.1% was fully biodegraded) (Table 5) [32,34]. This low removal efficiency (RE) was also observed for other phytoestrogens. For instance, the results of the endpoint “STP Total Removal” also showed inefficient removal (i.e., RE < 12%) of four other compounds, namely coumestrol (RE = 2.00%), enterolactone (RE = 3.95%), biochanin A (RE = 11.14%), and formononetin (RE = 6.74%) (Table 5) [32,34]. In light of these findings, it can be surmised that these six phytoestrogens may exhibit potential environmental persistence [32,34]. Only EV (RE = 93.71%) and 8-prenylnaringenin (RE = 76.73%) demonstrated effective removal in the CASTP (Table 5) [32,34].
Regarding the log KOW, coumestrol demonstrated a low bioaccumulation potential in soil, sediment, and sludge (i.e., log KOW < 2.5) (Table 5) [32,35]. Conversely, GEN, DAI, enterolactone, biochanin A, and formononetin demonstrated medium bioaccumulation potential (i.e., 2.5 < log KOW < 4.5) (Table 5) [32,35]. Only EV and 8-prenylnaringenin exhibited high bioaccumulation potential, with both compounds displaying a log KOW value exceeding 4.5 (Table 5) [32,35].
Ultimately, considering the potential ecological risks associated with these compounds, a prioritization procedure based on PBT criteria was employed (see Table 5). This approach was outlined by Roveri et al. (2022). The “T” criterion was identified as the most concerning among the entire range of phytoestrogens, with a total score of 33, followed by the “P” criterion, which scored 32. The criterion “B” (total score = 29) was identified as the lowest-risk compound in this final ranking (Table 5). Conversely, biochanin A and formononetin both ranked first and obtained a maximum individual score of 13 from the sum of the three criteria (Table 5; Figure 1). In the event of a tie between two phytoestrogens, the “B” criterion was employed to determine the ranking. Consequently, biochanin A (log Kow = 3.41) was assigned to the first rank, followed by formononetin (log Kow = 3.11) (Table 5; Figure 1). To determine the second and third positions, the same tiebreaker criterion was applied, with further details provided in Table 5 and Figure 1. Accordingly, the final ranking of the PBT approach was as follows: biochanin A (the highest priority), followed by formononetin, GEN, enterolactone, DAI, EV, coumestrol, and 8-prenylnaringenin (Table 5; Figure 1).

4. Conclusions

This study represents the initial phase in the evaluation of the adverse biological effects of two phytoestrogens (GEN and DAI) on aquatic organisms. The results revealed toxic effects for GEN and DAI at levels of a few mg/L for the tropical sea urchin E. lucunter. Both phytoestrogens were classified as “toxic” to E. lucunter. Regarding EV (the corresponding synthetic pharmaceutical selected for comparison purposes), the results showed a higher IC50 value than the results observed for the phytoestrogens. EV was classified as “harmful” following the same classification criteria [43] adopted for GEN and DAI.
Additionally, the RQ assessment results revealed the following trend: (i) 50% of the ecological risk assessments using GEN and DAI showed low toxicity to E. lucunter (0.01 ≤ RQ < 0.1). Therefore, both warrant attention in coastal waters. The risk assessment for EV could not be performed due to the absence of studies detecting this compound in coastal waters. The final ranking of the PBT approach in coastal waters was as follows: biochanin A (the highest priority), followed by formononetin, GEN, enterolactone, DAI, EV, coumestrol, and 8-prenylnaringenin.
In conclusion, these findings emphasize the necessity for the implementation of monitoring and reduction programs for these substances in sewage treatment plants on a global scale.

Author Contributions

B.F.L., R.B.K. and G.A.d.S.: Conceptualization, Data curation, Writing—original draft. V.R.: Methodology, Formal analysis; writing—review and editing; F.H.P. and F.S.C.: Technical support regarding the ecotoxicity analyses; W.T.: Review and editing; L.L.G.: Supervision, methodology, formal analysis, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available within the article.

Acknowledgments

BFL would like to thank CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for her research fellowship.

Conflicts of Interest

The authors declare no conflict of interest.

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Coasts 04 00034 g001
Figure 1. The final ranking of the prediction of persistence (P), bioaccumulation (B), and toxicity (T) of the five worldwide consumed phytoestrogens was selected for comparison with GEN, DAI, and EV. A five-point scale was employed to assign a rank to each criterion, with 5 indicating the highest level of concern and 1 indicating the lowest level of concern. The final ranking of the PBT was obtained by summing the ranks of the three criteria. In the event of a tie between the phytoestrogens in the final ranking, the “B” criterion was employed as a tiebreaker to determine the ranking (this criterion was applied to establish the order of the top three). The level of concern associated with phytoestrogens decreased in a left-to-right progression. The maximum score obtained by summing the four criteria was 13, indicating that biochanin A is the compound of primary concern. Conversely, 8-prenylnaringenin was positioned fourth, having received the lowest score of the ranking, namely 10 points. Further details on the PBT criterion can be found in Table 5 and Section 2.5.
Figure 1. The final ranking of the prediction of persistence (P), bioaccumulation (B), and toxicity (T) of the five worldwide consumed phytoestrogens was selected for comparison with GEN, DAI, and EV. A five-point scale was employed to assign a rank to each criterion, with 5 indicating the highest level of concern and 1 indicating the lowest level of concern. The final ranking of the PBT was obtained by summing the ranks of the three criteria. In the event of a tie between the phytoestrogens in the final ranking, the “B” criterion was employed as a tiebreaker to determine the ranking (this criterion was applied to establish the order of the top three). The level of concern associated with phytoestrogens decreased in a left-to-right progression. The maximum score obtained by summing the four criteria was 13, indicating that biochanin A is the compound of primary concern. Conversely, 8-prenylnaringenin was positioned fourth, having received the lowest score of the ranking, namely 10 points. Further details on the PBT criterion can be found in Table 5 and Section 2.5.
Coasts 04 00034 g001
Table 1. The following information is presented in tabular form: (i) Chemical Abstracts Service (CAS) number; (ii) molecular formula (MF); and (iii) SMILES (SM) strings of the seven most consumed phytoestrogens worldwide (including genistein and daidzein,) and estradiol valerate. The endpoints were obtained from the PubChem database. Moreover, the table demonstrates the following: (iv) The outcomes of “STP Total Removal” (%); (v) the outcomes of Log KOW (bioaccumulation); and (iv) the outcomes of chronic values (Chv). For ChV assessments, the lower toxicity value was assumed to assign the level of concern (values in bold). The “STP total removal”, the “log KOW”, and the “ChV” values of the phytoestrogens were determined utilizing EPI Suite software.
Table 1. The following information is presented in tabular form: (i) Chemical Abstracts Service (CAS) number; (ii) molecular formula (MF); and (iii) SMILES (SM) strings of the seven most consumed phytoestrogens worldwide (including genistein and daidzein,) and estradiol valerate. The endpoints were obtained from the PubChem database. Moreover, the table demonstrates the following: (iv) The outcomes of “STP Total Removal” (%); (v) the outcomes of Log KOW (bioaccumulation); and (iv) the outcomes of chronic values (Chv). For ChV assessments, the lower toxicity value was assumed to assign the level of concern (values in bold). The “STP total removal”, the “log KOW”, and the “ChV” values of the phytoestrogens were determined utilizing EPI Suite software.
QSARGeneral FeaturesSewage Treatment Plants
(STPs) Total Removal		BioaccumulationToxicity Effects
PubChem DatabaseEPI SuiteEPI Suite (ECOSAR)
CompoundCASMFSMSTP (%)Total Biodegradation (%)Total Sludge Adsorption (%)Log KOWChV (mg/L)
AlgaeCrustaceanFish
genistein446-72-0C15H10O5C1=CC(=CC=C1C2=COC3=CC(=CC(=C3C2=O)O)O)O4.540.114.422.840.44528802200
daidzein486-66-8C15H10O4C1=CC(=CC=C1C2=COC3=C(C2=O)C=CC(=C3)O)O3.250.103.142.550.50445123258
estradiol valerate979-32-8C23H32O3CCCCC(=O)OC1CCC2C1(CCC3C2CCC4=C3C=CC(=C4)O)C93.710.7792.546.420.0370.0310.004
8-prenylnaringenin53846-50-7C20H20O5CC(=CCC1=C2C(=C(C=C1O)O)C(=O)CC(O2)C3=CC=C(C=C3)O)C76.730.6776.064.970.1470.2350.109
coumestrol479-13-0C15H8O5C1=CC2=C(C=C1O)OC3=C2C(=O)OC4=C3C=CC(=C4)O2.000.091.901.570.9851072105243
enterolactone78473-71-9C18H18O4C1C(C(C(=O)O1)CC2=CC(=CC=C2)O)CC3=CC(=CC=C3)O3.950.113.842.730.52638400.958
biochanin A491-80-5C16H12O5COC1=CC=C(C=C1)C2=COC3=CC(=CC(=C3C2=O)O)O11.140.1710.973.410.32834090.984
formononetin485-72-3C16H12O4COC1=CC=C(C=C1)C2=COC3=C(C2=O)C=CC(=C3)O6.740.136.613.1129850.6270.795
Table 2. Criteria thresholds for the PBT ranking have been applied to seven of the most consumed phytoestrogens worldwide, including soy isoflavones such as genistein and daidzein, and estradiol valerate. For further details, please refer to M and M (Section 2.4).
Table 2. Criteria thresholds for the PBT ranking have been applied to seven of the most consumed phytoestrogens worldwide, including soy isoflavones such as genistein and daidzein, and estradiol valerate. For further details, please refer to M and M (Section 2.4).
Rank
(Criteria)		PBT
STP Removal
(%)		Log KowChV
(mg/L)
1≥80<1>100
2≥60≥1≤100
3≥40≥2≤10
4≥20≥3≤1
5<20≥4.5≤0.1
Table 3. Results of the embryo–larval assays (n = 4) of genistein, daidzein, and estradiol valerate (NOEC, LOEC, and IC50) on Echinometra lucunter (chronic toxicity test). Note: (i) IC50 (median inhibitory concentration) and confidence limits; (ii) NOEC (no observed effect concentration on the test organism); (iii) LOEC (lowest observed effect concentration that causes a statistically significant effect on the test organisms).
Table 3. Results of the embryo–larval assays (n = 4) of genistein, daidzein, and estradiol valerate (NOEC, LOEC, and IC50) on Echinometra lucunter (chronic toxicity test). Note: (i) IC50 (median inhibitory concentration) and confidence limits; (ii) NOEC (no observed effect concentration on the test organism); (iii) LOEC (lowest observed effect concentration that causes a statistically significant effect on the test organisms).
CompoundEchinometra lucunter (Embryo–Larval Development Assay)
IC50 (mg/L)NOEC (mg/L)LOEC (mg/L)
Genistein3.37 (3.01–4.02)<1.871.87
Daidzein2.60 (2.35–2.84)<1.871.87
Estradiol valerate28.40 (24.10–30.41)<6.256.25
Table 4. Results of the ecological risk assessment (RQ = MEC/PNEC) for the two phytoestrogens (genistein and daidzein). Furthermore, the table presents the following: (i) MEC values (in ng/L) obtained from peer-reviewed publications; (ii) PNEC values (in ng/L) derived from ecotoxicity experiments performed with the phytoestrogens in the present study. The PNEC values were obtained through a chronic ecotoxicological assay using a non-target seawater organism (the sea urchin Echinometra lucunter).
Table 4. Results of the ecological risk assessment (RQ = MEC/PNEC) for the two phytoestrogens (genistein and daidzein). Furthermore, the table presents the following: (i) MEC values (in ng/L) obtained from peer-reviewed publications; (ii) PNEC values (in ng/L) derived from ecotoxicity experiments performed with the phytoestrogens in the present study. The PNEC values were obtained through a chronic ecotoxicological assay using a non-target seawater organism (the sea urchin Echinometra lucunter).
Worldwide Peer-Reviewed Publications (MEC)Toxicity Experiments Performed with the Phytoestrogens in the Present Study (PNEC)MEC/PNEC
PhytoestrogenMEC (ng/L)MatrixReferenceOrganisms/SpeciesEndpointConcentrations (ng/L)AFPNEC (ng/L)ReferenceRQ
Genistein0.61Seawater (Baltic Sea)[47]Echinometra lucunterNOEC1,870,00010018,700This study0.00
Genistein826.0Mondego River estuary (Portugal)[48]Echinometra lucunterNOEC1,870,00010018,700This study0.04
Genistein135.0Douro River estuary (Portugal)[10]Echinometra lucunterNOEC1,870,00010018,700This study0.01
Genistein64.3Coastal Lagoon (Portugal)[49]Echinometra lucunterNOEC1,870,00010018,700This study0.00
Genistein12.9Pearl River estuary (China)[50]Echinometra lucunterNOEC1,870,00010018,700This study0.00
Daidzein0.43Seawater (Baltic Sea)[47]Echinometra lucunterNOEC1,870,00010018,700This study0.00
Daidzein270.0Mondego River estuary (Portugal)[48]Echinometra lucunterNOEC1,870,00010018,700This study0.01
Daidzein277.4Douro River estuary (Portugal)[10]Echinometra lucunterNOEC1,870,00010018,700This study0.01
Daidzein147.0Coastal Lagoon (Portugal)[49]Echinometra lucunterNOEC1,870,00010018,700This study0.01
Daidzein1.9Pearl River estuary (South)[50] Echinometra lucunterNOEC1,870,00010018,700This study0.00
Note: (i) MEC: measured environmental concentration; (ii) NOEC: no observed effect concentration; (iii) AF: assessment factor; (iv) PNEC: predicted no-effect concentration; (v) risk quotient (RQ). In the last column, risk quotients (RQ) for chronic tests (i.e., without risk, signaled in white; low risk, signaled in green). For further details, please see Section 2.4.
Table 5. Ranking of the persistence (“P”: based on the endpoint “STP—wastewater treatment plant total removal”; expressed in percentage of removal (%)), bioaccumulation (“B”: based on log Kow), and toxicity (“T”: based on chronic values (ChV; expressed in mg/L), (PBT criterion) of the seven most consumed phytoestrogens worldwide (including soy isoflavone, genistein, and daidzein) and estradiol valerate. Each criterion was assigned five ranks (levels of concern), from “5” (highest level of concern) to “1” (lowest level of concern). The final PBT score was then obtained by summing the ranks of the three criteria. For more details on the PBT criteria, see Table 2 and Section 2.5.
Table 5. Ranking of the persistence (“P”: based on the endpoint “STP—wastewater treatment plant total removal”; expressed in percentage of removal (%)), bioaccumulation (“B”: based on log Kow), and toxicity (“T”: based on chronic values (ChV; expressed in mg/L), (PBT criterion) of the seven most consumed phytoestrogens worldwide (including soy isoflavone, genistein, and daidzein) and estradiol valerate. Each criterion was assigned five ranks (levels of concern), from “5” (highest level of concern) to “1” (lowest level of concern). The final PBT score was then obtained by summing the ranks of the three criteria. For more details on the PBT criteria, see Table 2 and Section 2.5.
Compound1st Level of ConcernPersistence
(STP Total Removal)		2nd Level of ConcernBioaccumulation
(Log Kow)		3rd Level of ConcernToxicity
(ChV; mg/L)		Sum of Three Levels of ConcernFinal Ranking
Genistein54.5432.8440.445122nd
Daidzein53.2532.5540.504122nd
Estradiol valerate193.7156.4250.004113rd
8-prenylnaringenin176.7354.9740.109104th
Coumestrol52.0021.5740.985113rd
Enterolactone53.9532.7340.526122nd
Biochanin A511.1443.4140.328131st
Formononetin56.7443.1140.627131st
Concern level summed32
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Guimarães, L.L.; Lourenço, B.F.; Pusceddu, F.H.; Cortez, F.S.; Kiyotani, R.B.; dos Santos, G.A.; Toma, W.; Roveri, V. Predicting the Ecological Risks of Phytoestrogens in Coastal Waters Using In Silico and In Vitro Approaches. Coasts 2024, 4, 651-666. https://doi.org/10.3390/coasts4040034

AMA Style

Guimarães LL, Lourenço BF, Pusceddu FH, Cortez FS, Kiyotani RB, dos Santos GA, Toma W, Roveri V. Predicting the Ecological Risks of Phytoestrogens in Coastal Waters Using In Silico and In Vitro Approaches. Coasts. 2024; 4(4):651-666. https://doi.org/10.3390/coasts4040034

Chicago/Turabian Style

Guimarães, Luciana Lopes, Bárbara Faria Lourenço, Fabio Hermes Pusceddu, Fernando Sanzi Cortez, Rafael Barreiros Kiyotani, Gilmar Aparecido dos Santos, Walber Toma, and Vinicius Roveri. 2024. "Predicting the Ecological Risks of Phytoestrogens in Coastal Waters Using In Silico and In Vitro Approaches" Coasts 4, no. 4: 651-666. https://doi.org/10.3390/coasts4040034

APA Style

Guimarães, L. L., Lourenço, B. F., Pusceddu, F. H., Cortez, F. S., Kiyotani, R. B., dos Santos, G. A., Toma, W., & Roveri, V. (2024). Predicting the Ecological Risks of Phytoestrogens in Coastal Waters Using In Silico and In Vitro Approaches. Coasts, 4(4), 651-666. https://doi.org/10.3390/coasts4040034

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