Comprehensive Study on Endocrine Disruptor Removal from Wastewater Using Different Microalgae Species

Abstract

The concentration of endocrine disruptor compounds (EDCs) in wastewater is increasing, posing significant risks to living organisms. This study concerns the simultaneous degradation of a variety of EDCs from wastewater, including methylparaben (MeP), propylparaben (PrP), butylparaben (BuP), benzophenone (BP), bisphenol A (BPA), and estrone (E), in the presence of the microalgae Scenedesmus sp. or Chlorella vulgaris. The potential for the abiotic removal of these EDCs and their underlying degradation mechanisms were also studied. The presence of microalgae significantly enhanced the degradation of parabens, achieving complete removal within 7 days, primarily through the mechanism of biodegradation. BPA removal was also improved by microalgae, reaching 82% and 90% within 7 days with Scenedesmus sp. and C. vulgaris, respectively. BP degradation was predominantly abiotic, accomplishing 95% removal in 7 days. E degradation was mainly abiotic, achieving approximately 40% within 7 days, with a notable contribution from a biodegradation mechanism in the later stages, accounting for 27% and 40% of the final total removal in the presence of Scenedesmus sp. and C. vulgaris, respectively. This study provides insights into the mechanisms of EDC degradation by microalgae, highlighting the potential of Scenedesmus sp. and C. vulgaris to remove a mixture of EDCs from wastewater.

1. Introduction

Endocrine disruptor compounds (EDCs) are organic chemicals that interfere with the proper functionality of the endocrine system, causing adverse health effects in organisms and their populations [1]. This group of compounds is highly heterogeneous and can be classified as natural or synthetic EDCs. On the one hand, natural EDCs can contain some heavy metals or substances, like phytoestrogens and cyanotoxins, derived from living beings such as plants or bacteria [2]. On the other hand, synthetic EDCs are artificial compounds mostly used for industrial or agricultural purposes, such as pesticides, personal care products (PCPs), or plasticisers [3]. Currently, synthetic EDCs, including parabens, benzophenone and its derivatives, bisphenols, and oestrogens, are the most common endocrine disruptors due to their extensive production and use [4].

According to the literature [5,6,7], parabens, such as methylparaben (MeP), propylparaben (PrP), butylparaben (BuP), benzophenone (BP), bisphenol A (BPA), and estrone (E), are among the most widespread EDCs found in urban wastewater because they are present in everyday products. MeP, PrP, and BuP belong to p-hydroxybenzoic acid (PHBA) alkyl ester derivatives. Parabens are used as preservatives in food, pharmaceuticals, and PCPs due to their antibacterial and antifungal properties [8]. The adverse effects of parabens on human health may increase the proliferation of specific cancer cells [9], such as in breast cell cancer [10]. They can induce phenotype transformation on non-transformed human breast epithelial cells [11]. Additionally, paraben exposure can disrupt thyroid hormones, including thyroxine and triiodothyronine [12]. BP and its derivatives are the most-used UV filters for sunscreen products, skin creams, cosmetics, hair sprays, body lotions, hair dyes, shampoos, and other PCPs [13]. Several studies have investigated the potential health risks associated with benzophenones [14]. For example, a positive correlation between BP-3 urinary concentration and osteoarthritis was reported [15], and exposure to a high BP-1 concentration may be associated with endometriosis [16]. BPA is a diphenylmethane derivative, an organic synthetic compound used as an additive in producing polycarbonate plastics and epoxy resins [17]. Concerning the adverse effects of BPA on human health, higher urinary levels of this compound are positively related to diabetes mellitus in adults [18], coronary heart diseases in the adult population [19], and obesity in children and adolescents [20]. Finally, E is a naturally occurring hormone belonging to the oestrogen family. It is principally produced by the ovaries, adipose tissue, fibroblasts, skin, placenta, and brain [21]. The increasing consumption of hormones from contraceptive drugs and hormonal therapy in postmenopausal women is related to elevated levels of estrone in wastewater [22]. This fact represents a considerable risk, as E contamination could facilitate processes such as feminisation, reproductive dysfunction, and carcinogenesis in the surrounding environment [23].

These synthetic EDCs are widely distributed throughout the environment, with their highest concentration in water, though they are also present in air, soil, sludge, and sediments [24,25,26,27,28]. The principal sources of surface water contamination are industrial and urban sewage discharge effluents [29]. Conventional wastewater treatment plants, WWTPs, are usually ineffective at removing EDCs [30]. The typical concentration ranges of these pollutants in WWTP influents are as follows: 41.1–2466 ng/L of MeP, 47.4–5700 ng/L of PrP, 7.05–1602 ng/L of BuP [31], 321–951 ng/L of BP [13], 46.4–986 ng/L of BPA [32], and 49–98.9 ng/L of E [33,34]. Various processes have been investigated for removing these EDCs, including adsorption by activated carbon, chemical advanced oxidation, chemical precipitation, and ozonation [35]. However, these treatments have drawbacks, such as low efficiencies when applied to real wastewater and high operational costs [36]. In this context, microalgae provide a promising solution for EDC elimination.

Microalgae are photosynthetic, free-floating microorganisms capable of forming filaments and colonies. They exhibit a remarkable ability to adapt to extreme ecological habitats. Through cellular activities, microalgae convert light and carbon dioxide (CO₂) into various specialised chemicals, including carbohydrates, proteins, lipids, vitamins, and pigments [37]. Compared to the previously mentioned processes, the microalgae-based removal of EDCs offers distinct advantages, including the ability to fix CO₂ [38] and the removal of nutrients from contaminated water [39]. Moreover, the biomass produced can be further processed for other uses, such as energy production [40]. The removal of EDCs using photosynthetic microorganisms can be driven through four main mechanisms: bioadsorption, bioaccumulation, biodegradation, and photodegradation [41]. The most crucial step in the bioremediation process is the selection of microalgae, as their removal capacity can be influenced by heavy metals or other contaminants in wastewater [42]. Although various genera can remove EDCs from wastewater, Chlorella and Scenedesmus are the most frequently reported in the literature [43,44,45,46,47,48]. Another advantage of these two microalgae is that they can be isolated from WWTPs [49,50,51]. The existing literature includes several examples demonstrating the effectiveness of microalgae in removing EDCs. For instance, Scenedesmus obliquus and C. vulgaris have been shown to achieve removal rates of 99% for BPA, 87% for E, and 100% for both MeP and PrP within 7 days [52]. Additionally, S. obliquus biodegraded 96.66 and 74.38% of 0.5 and 2 mg of BP-3/L, respectively, in 10 days [53]. Other studies have evaluated the elimination of EDCs using a consortium with different microalgae and cyanobacteria, such as Anabaena cylindrica, Chlorococcus, Spirulina platensis, Chlorella, Scenedesmus quadricauda, and Anabaena sp., achieving approximately 80% removal within 6 days in an algae pond system [54]. Furthermore, Chlamydomonas mexicana and C. vulgaris can biodegrade 40% of BPA in 10 days [55]. However, all these studies primarily focus on eliminating one or two specific compounds or micropollutants within the same family, often with similar chemical structures [48,56,57,58]. Consequently, there is still a lack of research on the simultaneous removal of mixtures of EDCs from different families using these microorganisms. Thus, the novelty of our work is aligned with this challenge as it is focused on developing a microalgae-based system for the simultaneous removal of EDCs from different families, aiming to provide new insights into this field, including the study of EDC fate.

In this context, C. vulgaris and Scenedesmus sp. were cultivated to simultaneously remove EDCs (MeP, PrP, BuP, BP, BPA, and E) and nutrients from wastewater. The proposed system utilises microalgae to treat effluent from primary decantation, enabling the simultaneous removal of both nutrients and EDCs. A concentration of 1 ppm of each EDC was selected based on the values reported in the literature for EDC removal [43,59,60,61]. Additionally, this concentration provides a wide working range for quantifying the EDCs. This study includes the determination of the main degradation pathways of each EDC (abiotic, bioadsorption, bioaccumulation, and biodegradation).

2. Materials and Methods

2.1. Chemicals and Reagents

MeP (CAS Number 99-76-3), PrP (CAS Number 94-13-3), BuP (CAS Number 94-26-8), BP (CAS Number 119-61-9), BPA (CAS Number 80-05-7), and E (CAS Number 53-16-7, purity ≥ 99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). LC-MS grade methanol and HPLC grade acetonitrile were acquired from Scharlau (Barcelona, Spain). Mili-Q and distilled water were produced in Autwomatic plus 1+2 (Wasserlab, Barbatáin, Spain). Salts (NaCl, CaCl₂, MgSO₄·7H₂O, KH₂PO₄, and NH₄Cl) used in synthetic wastewater (SWW) preparation were acquired from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions for each EDC were prepared in methanol (1000 mg/L) and stored in darkness in a refrigerator at 4 °C. An aliquot of these stock solutions was diluted in SWW to achieve a final concentration of 10 ppm for each pollutant. This diluted solution spiked all doped samples, which were then kept refrigerated as stock solutions.

2.2. Experiments

EDC biodegradation was evaluated in the presence of microalgae, light, and aeration (LOM). On the other hand, the abiotic removal of EDCs was assessed through control experiments conducted using light (L), aeration (O), and their combination (LO). All experiments were performed in 50 mL batch reactors, with a working volume of 48 mL, over 7 days.

2.2.1. LOM Experiments

C. vulgaris and Scenedesmus sp. were the selected microalgae species, and the inoculums were acquired from the strain collection of Banco Español de Algas (Las Palmas de Gran Canarias, Spain). The SWW used for microalgae cultivation was prepared according to the composition of a typical effluent from the primary decantation of an urban WWTP [62]: 7 mg NaCl, 4 mg CaCl₂, 2 mg MgSO₄·7H₂O, 15 mg KH₂PO₄, and 115.6 mg NH₄Cl per litre of deionised water. Reactors in the LOM experiments were loaded with a combination of SWW and microalgae inoculum to achieve an initial biomass concentration of 0.1 g/L. Every reactor was spiked with 1 ppm of each EDC from a 10 ppm solution. Control experiments without contaminants were also performed. All experiments were conducted under continuous aeration using air pumps, and the air was pre-filtered through 0.45 µm nylon membranes before reaching the cultures (Agilent Technologies, Santa Clara, CA, USA). Reactors were additionally irradiated using 12 V LED strips, offering an irradiance of 108 µmol·foton·m⁻²·s⁻¹. The photoperiod was established as 12 h of light followed by 12 h of darkness. This approach was cost-effective and sustainable, ensuring the health and resilience of the microalgae for consistent and stable pollutant-removal performance. Biomass growth was monitored daily. Dry cell weight (DCW) was determined by filtering 12 mL of microalgae culture through a pre-weighed nylon membrane (GVS, Bologna, Italy) and drying it at 56 °C for one week. The dried filters were weighed, and the DCW was calculated using a weight balance. Additionally, pH was measured daily using a pH meter (XS Instruments, Modena, Italy).

2.2.2. Abiotic Experiments

Reactors for L, O, and LO experiments were filled with SWW and spiked with 4.8 mL of 10 ppm EDC solution, achieving a final concentration of 1 ppm. Experiments L and LO were carried out under light exposure, following the procedure used for LOM runs. Conversely, O experiments were conducted under dark conditions with reactors wrapped with aluminium foil and kept without light. O and LO experiments were conducted under the same aeration conditions as LOM assays.

2.3. Supernatant Analysis

The concentration of EDCs in the supernatant was analysed daily for all samples using high-performance liquid chromatography (HPLC) (Agilent Technologies, Santa Clara, CA, USA) equipped with a DAD detector (Agilent Technologies, model 1260 DAD WR) and an Ascentis Express C18 column (100 mm × 4.6 mm, 5 µm, Supelco, Merck, Darmstadt, Germany). The mobile phase consisted of acetonitrile (with 0.1% formic acid) and water in a ratio of 30:70 for MeP and 40:60 for the rest of the EDCs detection. The flow rate was set to 1 mL/min. The detection wavelengths were established at 254 nm for parabens and BP and 210 nm for BPA and E. The signal-to-noise method was used to determine the limit of detection (LOD) and limit of quantification (LOQ). A signal–noise (S/N) ratio of three was adopted for LOD determination, and a S/N ratio of ten was selected for LOQ [63]. The results are presented in the Supplementary Material, along with the calibration curves and the working concentration ranges (Table S1).

The samples from the LOM experiments were centrifuged at 3500 rpm for 20 min. The supernatant was separated from the microalga cell pellets and filtered using a 0.45 μm nylon membrane. The samples were concentrated with solid-phase extraction (SPE), using 500 mg Extrabond C18 cartridges (Scharlau, Barcelona, Spain), and were sequentially preconditioned with methanol and water. The analytes were finally eluted with 2 mL of methanol, and 10 µL of the eluent was injected for the HPLC analysis previously mentioned. The concentrations of ammonium and phosphates were determined with 100,683 and 100,798 Spectroquant commercial kits, respectively (Merck, Darmstadt, Germany).

Supernatants from L, O, and LO experiments were filtered through 0.45 µm nylon membrane, and subsequently, EDCs were extracted and analysed by HPLC, as described for LOM experiments.

2.4. Mass Balance

A comprehensive study on the removal mechanisms of EDCs by microalgae was conducted to assess the contribution of bioadsorption, bioaccumulation, and biodegradation pathways. The biodegradation percentage of each EDC can be calculated as follows:

$$P\left(\%\right)=\left(A_t-A_r-A_d-A_a-A_c\right)·{\displaystyle \frac{100}{A_t}}$$

(1)

where A_t is the initial amount of each EDC added to the medium (μg at day 0). A_r is the amount of EDCs in the supernatant in the LOM experiments. A_d is the amount of EDCs adsorbed on the microalgal cell wall. As mentioned above, the samples from the LOM experiments were centrifuged, and the microalgae cells were harvested and separated from the supernatant. This biomass was washed with 1 mL of water and centrifuged at 3500 rpm for 20 min (model 5810, Eppendorf, Hamburg, Germany). The wash water was discarded, and cell pellets were resuspended in 1 mL of methanol. Finally, the pellets were sonicated for 15 min (37 kHz, 820 W) in an Elmasonic P bath (Elma Schmidbauer, Singen, Germany) to release the EDCs adsorbed on the cell wall into the methanol. The mixture was then centrifuged again, and the biomass was separated from the organic solvent containing the adsorbed EDCs. These samples were used to quantify the concentration of adsorbed EDCs using previously established HPLC methods. Finally, A_c is the amount of EDCs bioaccumulated inside the microalgal cells. To determine this value, 1 mL of methanol was added to the harvested cell pellet after the last centrifugation, and the solution was kept under stirring at 900 rpm overnight. To ensure cell wall lysis, the samples were sonicated for 1 h and then centrifuged at 3500 rpm for 20 min. The concentration of EDCs bioaccumulated by the microalgae was determined from the resulting supernatant using HPLC. Finally, A_a is the amount of EDCs removed by abiotic mechanisms. The results of the supernatant analysis in the LO experiments were used to estimate the removal fraction of the EDCs by abiotic processes.

The kinetic constants for MeP, PrP, BuP, BP, BPA, and E removal were also calculated for the O, LO, and LOM experiments using a pseudo-first-order model as follows:

$$Ln{C}_t=-kt+Ln{C}_0$$

(2)

where C₀ is the initial concentration (ppb) of each pollutant at day 0, C_t is the EDC concentration (ppb) at time t, k is the removal rate constant (h⁻¹), and t is the reaction time (h).

2.5. Statistical Analyses

All experiments in this study were conducted in triplicate to check their repeatability. A t-test was used to evaluate the differences between microalgae growth in the presence or absence of EDCs and possible discrepancies between abiotic and biodegradation EDC removal. Differences were considered significant when p < 0.05.

3. Results and Discussions

3.1. Microalgae Growth in the Presence of EDCs

Scenedesmus sp. and C. vulgaris were grown separately in the presence (doped) or absence (control) of an EDC mix for 7 days in SWW (LOM experiments), and the DCW was evaluated. Figure 1a shows how both microalgae species can grow in culture media with ammonium as a nitrogen source. C. vulgaris demonstrated superior adaptation to the SWW compared to Scenedesmus sp., resulting in an algal density of 0.55 and 0.67 g/L within 7 days for the control and doped samples, respectively. On the other hand, Scenedesmus sp. reached only 0.34 g/L and 0.29 g/L. C. vulgaris preferred ammonium as its optimal nitrogen source, even when both ammonium and nitrate are present in the culture media [64]. In contrast, Scenedesmus sp. exhibited lower algal density when ammonium was used as a nitrogen source compared to nitrate or urea [65]. Similar growth for both species was observed in the absence and presence of EDCs. A paired-samples t-test revealed that the presence of EDCs did not significantly impact microalgae growth (p-value > 0.05), except on day 1 for both species and day 7 for C. vulgaris (p-value < 0.05), where EDC presence enhanced growth. This fact has also been reported in previous studies, suggesting that microalgae growth is not negatively affected by the presence of EDCs. Instead, microalgae may adapt to low doses of these compounds in culture media, potentially enhancing their growth. For instance, the microalgae Tetraselmis sp. demonstrated adaptation to EDCs in the culture media with 1 ppm of BPA and other EDCs, showing no adverse effects on cell growth [66]. Additionally, the growth of C. vulgaris could be enhanced in the presence of MeP concentrations ranging from 1 to 5 ppm [67].

As observed in Figure 1b, the pH of the Scenedesmus sp. culture in the presence of EDCs continuously decays up to a pH value of approximately 4 after 7 days. These acid conditions can be attributed to the release of H⁺ when ammonium is the nitrogen source [68]. The optimal pH range for Scenedesmus sp. was between 8.5 and 6.5, achieved on day 3 (Figure 1b), which coincided with a decrease in biomass growth, as shown in Figure 1a [69]. Other authors observed this fact, suggesting that the reduction in pH has an inhibitory effect on Scenedesmus sp. growth. This inhibition is primarily due to acidic conditions acting as enzyme photosynthesis inhibitors. Consequently, the disruption of photosystem II reactions resulted in a lower algal cell density [70].

The optimal pH for C. vulgaris ranged between 6.0 and 9.0 [71]. Figure 1b shows a slight acidification trend for the C. vulgaris culture up to a pH of 7. The highest growth rates were observed from day 4 to day 7, as depicted in Figure 1a. This increase in algal growth led to elevated algal photosynthetic activity. Some studies suggest that higher photosynthesis rates can lead to a rise in pH, thus preventing the acidification of the culture media [72]. This phenomenon, therefore, was observed in the growth of C. vulgaris in the presence of EDCs and explains the high growth rate achieved with this microalga.

3.2. Nutrient Removal

Figure 2 shows the ammonium and phosphate removal by Scenedesmus sp. (a) and C. vulgaris (b) in the presence of EDCs for 7 days (LOM experiments). Figure 2a exhibits a complete phosphate depletion within 4 days of Scenedesmus sp. cultivation. However, only 32% of the ammonium was removed in 7 days. This low ammonium removal efficiency is associated with this microalga’s limited growth. Thus, the simultaneous complete removal of phosphate restricted ammonium consumption, which has also been reported in previous studies. For example, Scenedesmus sp. LX1 will completely remove (100%) the total phosphorus (TP) regardless of the N/P ratio, while the removal of total nitrogen (TN) strongly depends on that nutrient ratio [73]. A similar trend was observed when Scenedesmus sp. was cultivated under phosphorous-starved conditions. In this study, complete phosphorus depletion occurred within 11 days, but nitrogen removal remained negligible beyond this point [74].

Figure 2b shows the complete ammonium and phosphate removal in 7 days by C. vulgaris. This result agrees well with other reports in the literature. In this sense, C. sorokiniana completely depleted both ammonium and phosphorus in 7 days when the initial nitrogen concentration ranged from 20 to 40 mg N/L [75]. In addition, C. vulgaris cultivated in different anaerobic digestion effluents exhibited a nearly 100% removal of ammonium and phosphates within 10 days with an initial ammonium concentration of 40 mg N/L [76]. This total nutrient removal achieved during the cultivation of C. vulgaris in the presence of EDCs provides promising insights for the future development of WWTP biological treatments based on microalgae.

3.3. EDC Removal

The influence of light, oxygen, and the presence of microalgae on EDC removal rates was studied for each contaminant. As shown in Figure 3a–c, parabens do not exhibit a representative abiotic degradation (LO and O experiments). However, the presence of microalgae (LOM experiments) enhanced the removal rates of these compounds, resulting in complete elimination within 7 days for both microalga species, exhibiting excellent performance for the degradation of these contaminants. These results agree with those observed by other authors. In this sense, a high MeP degradation efficiency using C. vulgaris was reported, achieving the removal of 1 ppm of MeP within 2 days [67]. Furthermore, the complete removal of MeP and PrP, starting at 47.4 and 3.8 ng/L, respectively, was achieved within 7 days using C. reinhardtii, S. obliquus, C. pyrenoidosa, and C. vulgaris, separately [52].

Regarding BP degradation, similar high removal efficiencies were observed regardless of the type of experiment, as shown in Figure 3d. Thus, the efficiencies of BP removal in the LOM experiments on day 7 were 86% and 62% for Scenedesmus sp. and C. vulgaris, respectively, slightly lower than the ones obtained in the O and LO experiments (91% and 95%, respectively). These results suggest that the primary degradation pathway for this compound is most likely abiotic. No differences in removal rates were observed when comparing the O and LO experiments. Therefore, light does not significantly affect the elimination of BP, reaching only 20% removal within 7 days, as demonstrated in the Supplementary Material Section (Figure S1), which presents the results of the L experiments. Consequently, BP degradation may involve oxidation due to the presence of oxygen. Previous studies propose that the principal reactive oxygen species (ROS) involved in the photo-transformation of BP–3 are hydroxyl radicals (OH·) and superoxide radicals (·O₂) [77]. In addition, other research hypothesised that BP may be degraded naturally when it is present in water in low concentrations [78].

BPA degradation is shown in Figure 3e. This contaminant was removed in abiotic conditions (O and LO experiments) by 20%, which agrees with the results previously reported [55]. In addition, the photodegradation (L experiment) of BPA was negligible, as depicted in Figure S1 (Supplementary Material). BPA removal increased significantly from the first day in the LOM experiment with Scenedesmus sp. In contrast, significant BPA elimination in the LOM experiment with C. vulgaris was observed from day 4 onwards, coinciding with its exponential growth phase and the decrease in ammonium and phosphate levels, as shown in Figure 1a and Figure 2b, respectively. By the end of the 7 days, both Scenedesmus sp. and C. vulgaris achieved high BPA removal efficiencies, reaching 82% and 90%, respectively. These results are aligned with previous studies, such as the removal rate of 96% for BPA within 90 h using a consortium of Chlorophyceae-class microalgae and cyanobacteria, using a starting initial BPA concentration of 10 ppb [79]. On the other hand, Li et al. (2009) reported a degradation of BPA of nearly 92% within 16 days using Stephanodiscus hantzschii in a medium containing 1 ppm of this contaminant [80].

When analysing E removal, similar degradation values to those obtained for BPA (by 20%) were observed under abiotic conditions (O and LO experiments) during the first three days, as depicted in Figure 3f. However, abiotic degradation became more significant from day 4 to 7. As shown in Figure S1 (Supplementary Material), light was not a substantial factor in E removal. BP and E were the only compounds that exhibited abiotic degradation, so the potential synergistic effects between these pollutants were studied. For this purpose, the LO experiments were conducted without BP or E, keeping the operating conditions and the rest of the ECDs. The results indicated that E did not undergo abiotic degradation under the LO conditions without BP (Supplementary Material, Figure S2b). However, BP exhibited a similar degradation trend in the culture media with or without E, with the degradation rate being even higher in the presence of E at day 7 (95% compared to 60%, respectively) (Supplementary Material, Figure S2a). These findings suggest a positive synergistic effect on the degradation of BP and E when both compounds are present in the mixture. On the other hand, when C. vulgaris was included, a dramatic increase in removal efficiency was observed from day 4 to day 7, achieving a reduction in E of 83% within 7 days (Figure 3f).

Conversely, the presence of Scenedesmus sp. did not increase the percentage of E removed (70% in 7 days) compared to the O experiments (63%), although it was higher than in the LO experiments (40%). During their growth, microalgae produce algal extracellular organic matter (AEOM) primarily consisting of biopolymers such as polysaccharides and proteinaceous substances. AEOM plays a significant role in promoting the photochemical generation of short-lived radicals, including excited triplet-state dissolved organic matter (³DOM*), singlet oxygen (¹O₂), and hydroxyl radicals (·OH). These radicals contribute to the degradation of pollutants such as E [81]. Studies have indicated that C. vulgaris produces more AEOM than Scenedesmus quadricauda, potentially enhancing the photocatalytic degradation of E [82]. Notably, the high removal efficiencies of E achieved by C. vulgaris between days 4 and 7 (Figure 3f) align with its exponential growth phase observed in Figure 1a. This phase is characterised by rapid metabolic activity and increased AEOM release [81].

The differences in AEOM release could explain the variation in the final E removal between C. vulgaris and Scenedesmus sp. These removal results are aligned with previous research. For instance, a 79% reduction in E concentration was reported using Haematococcus pluvialis, Selenastrum capricornutum, and Scenedesmus quadricauda individually over 10 days [83]. Additionally, an overall E removal of 91% with S. obliquus and 52% with C. vulgaris in 5 days was demonstrated [84]. In that same study, Scenedesmus achieved a higher removal efficiency compared to C. vulgaris, which contrasts our findings. This discrepancy could be attributed to differences in cultivation methods and in the composition of the synthetic wastewater used. For instance, this other study employed a continuous cultivation mode, where reactors were fed daily, providing a more stable environment and consistent nutrient supply, potentially enhancing the performance of Scenedesmus over C. vulgaris.

Finally, the final removal percentages obtained for both microalgae after 7 days are presented in

Table 1. Final removal percentage rates at day 7 with Scenedesmus sp. and C. vulgaris and comparison with literature review. Errors are expressed as ± standard error of the mean (n = 3).

Microalgae	MeP	PrP	BuP	BP	BPA	E	Ref.
Scenedesmus sp.	100.0 ± 0.0%	99.4 ± 1.1%	99.2 ± 1.5%	85.6 ± 9.0%	81.8 ± 6.3%	67.0 ± 0.6%	This study
C. vulgaris	100.0 ± 0.0%	100.0 ± 0.0%	100.0 ± 0.0%	62.1 ± 9.1%	89.9 ± 2.0%	83.0 ± 2.0%
C. reinhardtii	100.0%	100.0%					[52]
S. obliquus	100.0%	100.0%
C. pyrenoidosa	100.0%	100.0%
C. vulgaris	100.0%	100.0%
Tetradesmus obliquus, C. vulgaris, Pseudanabaena sp., Scenedesmus sp. and Nitzscha sp.		89.0%					[85]
C. vulgaris	33.0–14.0%						[59]
S. obliquus				23.3–28.5% 1			[53]
Chlamydomonas reinhardtii				58.4% 1			[86]
C. vulgaris				14.0% 2			[87]
Chlorella pyrenoidosa					20.0–43.0%		[88]
C. mexicana					39.0%		[55]
C. vulgaris					28.0%
S. obliquus						91.0%	[84]
C. vulgaris						52.0%

¹ BP-3; ² BP-4.

and compared with the previous literature. As explained previously, comparing these results with others reported in the literature is difficult due to the considerable influence of culture conditions, such as the presence of aeration and the composition of the culture media.

Regarding parabens, complete removal was achieved for both microalgae. These removal percentages are within the range described by other authors. As previously mentioned, the 100% elimination of MeP and PrP using different microalgae species was reported [52]. Additionally, a PrP removal rate of approximately 89% was achieved from an initial concentration of 300 ng PrP/L using an anoxic–aerobic photobioreactor with a consortium of Tetradesmus obliquus, C. vulgaris, Pseudanabaena sp., Scenedesmus sp., and Nitzscha sp. [85]. However, these studies employed lower initial concentrations than those reported in our work. When higher concentrations of parabens were studied, only 33% and 14% removal of MeP were obtained using C. vulgaris at initial concentrations of 0.8 and 8.0 mg MeP/L, respectively [59]. Given the scarcity of the literature on BP microalgae removal, our results will be compared with those for similar molecules such as BP-3 and BP-4. In all cases, the removal values obtained in our study are superior to those reported in previous research. S. obliquus removed 23.3–28.5% of BP-3 after 10 days of cultivation, ranging from 0.1 to 3 mg BP-3/L [53]. A maximum BP-3 removal of 58.4% was achieved at an initial concentration of 0.01 μg BP-3/L using the green alga Chlamydomonas reinhardtii within 10 days, with decreasing efficiency at higher pollutant concentrations [86]. A removal rate of 14% at 1 mg BP-4/L was reported also using C. vulgaris [87]. The final BPA removal rates presented in Table 1 are higher than those reported by previous authors. Removal rates of 20.0%, 46.4%, 42.9%, and 43.0% were achieved using Chlorella pyrenoidosa after 120 h of culture with BPA concentrations of 2.0, 4.0, 6.0, and 8.0 mg BPA/L [88]. Microalgae C. mexicana and C. vulgaris achieved 39% and 28% removal, respectively, in a medium containing 1 mg BPA/L within 10 days [55]. Finally, the results of E removal are consistent with the previously shown literature. S. obliquus and C. vulgaris removed 91% and 52% of E, respectively [84], and other studies found E removal rates higher than 80% [83]. In summary, the results reported in our work are very promising and demonstrate the potential of using Scenedesmus sp. and C. vulgaris to simultaneously remove a variety of EDCs from wastewater, achieving ECD removal percentages higher, in general, than those reported in the literature.

To quantify degradation rates for each contaminant, kinetic constants were calculated from the above removal curves, assuming a pseudo-first-order model in all cases, according to Equation (2). The values obtained (

Table 2. Removal rate constants (k) estimated for abiotic EDC degradation and biodegradation using first-order kinetics.

Compound	kO (h−1) (R2)	kLO (h−1) (R2)	kLOM (h−1) (R2)
			Scenedesmus sp.	C. vulgaris
MeP	1.3 × 10−3 (0.868)	1.1 × 10−3 (0.841)	9.0 × 10−2 (0.983)	3.8 × 10−1 (0.928)
PrP	7.0 × 10−4 (0.831)	1.8 × 10−3 (0.852)	5.7 × 10−2 (0.968)	7.4 × 10−2 (0.972)
BuP	2.9 × 10−3 (0.951)	2.8 × 10−3 (0.961)	5.4 × 10−2 (0.982)	7.5 × 10−2 (0.957)
BP	1.3 × 10−2 (0.936)	1.6 × 10−2 (0.958)	1.1 × 10−2 (0.924)	6.0 × 10−3 (0.901)
BPA	2.0 × 10−3 (0.940)	1.1 × 10−3 (0.956)	2.1 × 10−2 (0.976)	1.3 × 10−2 (0.815)
E	5.9 × 10−3 (0.966)	3.1 × 10−3 (0.954)	5.2 × 10−3 (0.819)	9.9 × 10−3 (0.805)

) indicate that the degradation rate is significantly higher when using microalgae compared to abiotic conditions, with C. vulgaris being superior to Scenedesmus sp. for paraben removal. For BP, the highest degradation rates were achieved under abiotic conditions, as explained above, reinforcing the hypothesis that the primary degradation pathway for this contaminant is driven in these conditions. Regarding BPA degradation, Table 2 shows that the rate constant values are higher when microalgae are present. This, along with the high removal efficiency values mentioned earlier, suggests the suitability of these species to degrade this compound. Finally, in the case of E removal, the rate constants were similar, obtaining higher values in the presence of microalgae.

3.4. Mass Balance

Figure 4, Figure 5, Figure 6 and Figure 7 depict the contribution of the different removal mechanisms (abiotic, biosorption, bioaccumulation, and biodegradation) for the studied contaminants within 7 days. The results were calculated using Equation (1) and expressed as the amount of contaminant removed concerning its initial concentration for each experiment.

The primary removal mechanism for parabens in both species was biodegradation (Figure 4a–f). However, Scenedesmus sp. also showed a low percentage of biosorption and bioaccumulation of MeP (Figure 4a) and PrP (Figure 4c). The adsorbed fraction exhibited a consistent trend for these compounds, initially increasing from day 1 to day 3 (from 0.43 to 1.71% for MeP and from 0.57 to 1.44% for PrP), and subsequently decreasing until day 7, reaching less than 0.04% for MeP and 0.64% for PrP. On the other hand, the bioaccumulated fraction of MeP increased during the initial days from 0.04–0.11% to 0.88% but showed an almost complete reduction (<0.04%) by day 7. Conversely, the bioaccumulated fraction of PrP showed an upward trend from day 1 to day 7, increasing from less than 0.005% to 0.58%. These results suggest that MeP and PrP presented a slow diffusion into the cells, and MeP probably underwent biodegradation by intracellular enzymes. The adsorbed and bioaccumulated fractions for BuP (Figure 4e) were negligible, accounting for less than 0.08% of the total contaminant concentration removed. The concentrations detected in both measurements were below the LOD.

The absence of parabens adsorbed by C. vulgaris can be observed by analysing the results for this species (Figure 4b,d,f). In this sense, previous studies have reported that MeP biosorption by this species is negligible due to the hydrophobicity of its cell wall [59]. This fact agrees with our results, which showed adsorbed fractions lower than 0.05% for MeP and PrP and ranging between 0.53 and 0.11% for BuP. Furthermore, the contribution of the bioaccumulation pathway to the removal of the studied parabens was also negligible. When EDCs are present inside microalgae cells, an oxidative stress defence mechanism may be triggered, leading to the formation of ROS in their organelles. According to the literature, no excess ROS formation was observed during the growth of C. vulgaris in the presence of 0.8 mg MeP/L over 7 days, reinforcing the theory that parabens do not diffuse into the cells of this species [59]. Thus, biodegradation appeared as the main pathway for paraben degradation in C. vulgaris.

BP exhibited a remarkable abiotic degradation pathway (Figure 5a,b). As previously explained, no significant differences were observed between the BP removal efficiency in the LO experiments (abiotic conditions) and the LOM experiments, regardless of the microalgae used, obtaining a p-value > 0.05 in both cases. In addition, BP showed low adsorption onto the cell wall, and this fraction was higher when using C. vulgaris (Figure 5b) compared to Scenedesmus sp. (Figure 5a). As can be observed in Figure 5a,b, the contribution of the bioaccumulation pathway was negligible for both microalgae species. In the case of Scenedesmus sp., values ranging between 0.09 and 0.52% (concentrations between LQD and LOD) were obtained from day 1 to day 7 for bioaccumulation. A similar trend was observed for C. vulgaris, its bioaccumulation percentages of BP ranging between less than 0.12% (concentrations below the LOD) and 0.53% from day 1 to 7, respectively. Comparing the obtained results with previous studies on unsubstituted BP is challenging due to the scarcity of such studies. The most similar compounds found in the literature were BP-3 and BP-4. In this way, Lee et al. (2020) achieved 97% biodegradation of 0.5 ppm of BP-3 within 10 days using S. obliquus, and approximately 3% attributed to abiotic removal [53]. Similarly, Huang et al. (2018) found negligible BP-4 adsorption and absorption onto C. vulgaris strains, with the concentration decreasing solely due to biodegradation, achieving 14% at 1 mgBP-4/L within 13 days [87]. The lower abiotic degradations observed in the literature compared to our research are attributed to differences in cultivation conditions. Specifically, other studies’ cultures were maintained only with agitation, without aeration, leading to the absence of oxygen-derived ROS in the culture medium, which promoted BP removal.

The main removal pathway for BPA using Scenedesmus sp. was biodegradation (Figure 6a). Adsorption accounted for less than 1% of the total removal during the two first days, decreasing to less than 0.06% in the following days. Additionally, BPA was not detected inside the cell, indicating that the bioaccumulated fraction was negligible, remaining below 0.06% (under the LOD). The contributions of biosorption and bioaccumulation to BPA removal using C. vulgaris (Figure 6b) were higher. Both pathways were similar during the first three days, with bioaccumulation becoming predominant from day 4 onwards. These results agree with other authors, such as Ben Ouada et al. (2018), who reported the abiotic removal of BPA by 19%, with the fractions removed by accumulation, adsorption, and biodegradation by 1.3%, 11.6%, and 40%, respectively, within 5 days using the alkaliphilic Chlorophyta Picocystis with an initial concentration of 25 mg BPA/L [89]. Similarly, the abiotic removal values of BPA were 15.0% with a biodegradation pathway contribution of 25% using C. mexicana and C. vulgaris, respectively, at the end of a 10-day experiment with initial concentrations of 1 mg BPA/L [55].

As can be observed in Figure 7a,b, abiotic degradation and biodegradation were the main pathways to remove E. Comparing Scenedesmus sp. (Figure 7a) and C. vulgaris (Figure 7b), it can be observed that E removal occurred at earlier stages in the former species than in the latter. In contrast, C. vulgaris only presented a biodegradation contribution on the last day. As previously explained, C. vulgaris potentially releases AEOM at the end of its exponential growth phase, which promotes E degradation. Comparing our results is challenging because E is one of the least-studied oestrogens in this context, with limited previous research studies available. Additionally, among the existing studies, E is often examined as a degradation product of 17β-estradiol. For example, Ruksrithong and Phattarapattamawong (2019) found that adsorption accounted for 10% of total E removal using S. obliquus and C. vulgaris, identifying biodegradation as the primary removal mechanism for both species. The biodegradation of E by S. obliquus was 77%, whereas C. vulgaris degraded only 38% within 5 days [84]. As explained earlier, these results are only partially aligned with ours, primarily due to differences in cultivation.

4. Conclusions

Scenedesmus sp. and C. vulgaris were used to remove various EDCs. The total removal ratios of parabens (MeP, PrP, and BuP) were higher in the presence of both microalgae, achieving 100% removal within one or two days. Abiotic removal was not significant in this process, with biodegradation being the primary mechanism, while adsorption and bioaccumulation were negligible. BP and E exhibited high abiotic degradation, likely induced by derived ROS. However, the biodegradation pathway contributed significantly to E removal for both Scenedesmus sp. and C. vulgaris, achieving 67% and 83% final degradation rates, respectively. Biodegradation was the primary degradation pathway for BPA combined with adsorption. The contribution of the latter pathway was higher in the case of C. vulgaris than in Scenedesmus sp., with final degradation percentages of 90% and 82%, respectively. Overall, the promising results obtained in this work are of interest in the field, as they demonstrate the suitability of Scenedesmus sp. and C. vulgaris for EDC removal. This was proven not only for a specific family of these contaminants but simultaneously for a variety with different characteristics, showing the potential application of these microalgae for EDC removal from wastewater. In addition, the findings reported in this work on the routes implied for EDC removal can be a benchmark for further studies on the metabolic pathways driven by microalgae in these processes. Finally, the results of this research can have future implications for the use of microalgae for wastewater treatment, serving as a starting point to scale up these processes.