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Article

Assessing the Efficiency of Microalgae in the Removal of Salicylic Acid from Contaminated Water: Insights from Zebrafish Embryo Toxicity Tests

by
Carla Escapa
1,2,
Ricardo N. Coimbra
2,3,
Moonis Ali Khan
4,
Teresa Neuparth
1,
Miguel Machado Santos
1,5,* and
Marta Otero
2,3,*
1
CIMAR/CIIMAR—Interdisciplinary Centre of Marine and Environmental Research, Endocrine Disruptors and Emerging Contaminants Group, University of Porto, Av. General Norton de Matos s/n, 4450-208 Matosinhos, Portugal
2
Departamento de Química y Física Aplicadas, Universidad de León, Campus de Vegazana s/n, 24071 León, Spain
3
Department of Environment and Planning, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal
4
Chemistry Department, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
5
FCUP—Department of Biology, Faculty of Sciences of the University of Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal
*
Authors to whom correspondence should be addressed.
Water 2024, 16(13), 1874; https://doi.org/10.3390/w16131874
Submission received: 19 May 2024 / Revised: 18 June 2024 / Accepted: 24 June 2024 / Published: 29 June 2024
(This article belongs to the Special Issue Novel Insights on Wastewater Treatment Processes for Sustainable Removal of Emerging Contaminants)
Browse Figures
Versions Notes

Abstract

:
Microalgae-based water treatments for the removal of different types of pollution have been gaining considerable attention during the last few decades. However, information about microalgae potential in the removal of pharmaceuticals and personal care products (PPCPs) and the ecotoxicological outcomes is still limited. Therefore, in this work, we aimed at investigating salicylic acid removal from water by three different microalgae strains, namely Chlorella sorokiniana, Chlorella vulgaris and Scenedesmus obliquus. For such a purpose, photobioreactors were operated under batch and semi-continuous mode. Apart from determining the reduction in the concentration of salicylic acid attained by each strain, we used zebrafish embryo bioassays to assess the efficiency of microalgae to reduce its toxicity effects. S. obliquus was the strain that achieved the most significant decrease in the concentration and toxic effects of salicylic acid. Indeed, S. obliquus was able to rescue mortality and reduce abnormalities at practically 100%. The efficiency of C. sorokiniana and, especially, that of C. vulgaris were not so remarkable, indicating that the removal of SA and its toxic effects from water by microalgae is markedly strain dependent. The obtained results proved the importance of considering toxic effects for a more comprehensive evaluation of microalgae efficiency in the removal of PPCPs in view of an adequate selection for water treatment.

1. Introduction

Pharmaceuticals, together with personal care products (PCPs), pesticides, industrial chemicals or microplastics, are part of the so-called group of contaminants of emerging concern (CECs) [1]. They are not commonly monitored or regulated but they have the potential to enter the environment and cause known or potential adverse ecological and human health effects. Concern about these effects has led to intensive research on the environmental pathways, fate, removal, and overall impact of these substances [2]. Among CECs, the environmental presence of pharmaceuticals and PCPs (PPCPs) is especially worrying because they may cause physiological undesired responses in non-targeted species [3]. Indeed, pharmaceuticals and cosmetic residues are the main sources of micropollutants in urban wastewater according to the text of the new Urban Wastewater Treatment Directive (UWWTD) [4], which was recently agreed upon by the European Parliament and is expected to come into force at the end of this year. To avoid the discharge of these pollutants in natural waters, this text points to the necessity of implementing an additional treatment (quaternary treatment) for their removal in plants that treat a load of over 150,000 population equivalent (p.e.), or 10,000 p.e., based on a risk assessment [4]. Thus, in the actual context, the importance of research on efficient and sustainable treatment strategies for removing PPCPs from water is evident [5].
Due to its wide utilization in dermatological, cosmetic, and pharmaceutical formulations, salicylic acid (SA) is among the most frequently detected PPCPs in surface waters [6]. SA possesses keratolytic, bacteriostatic, fungicidal, and photoprotective properties, so it is largely exploited for the topical dermatological treatment of warts, hyperkeratosis, psoriasis, or acne, among others [7]. Regarding cosmetic uses, SA is used in skin ointment formulations as a peeling agent [8] and in sunscreen preparations [9]. As a pharmaceutical, SA is a non-steroid anti-inflammatory drug (NSAID) and the precursor to acetylsalicylic acid, mostly known as aspirin, which is used to treat pain, fever, inflammation, migraines, and reduce the risk of major adverse cardiovascular events [10]. On the other hand, after consumption, SA is the active metabolite from aspirin. In his book dedicated to aspirin, Diarmuid Jeffreys described this medicine as “the most remarkable drug the world has ever seen”, as “one of the astonishing inventions in history” and “one of the most endurably successful commercial products of all time” [11]. In fact, more than approximately 35,000 metric tons of aspirin are annually produced and consumed in the world [11]. Despite its widespread use, SA may cause acute and chronic toxicity known as salicylism, the symptoms of which include nausea, vomiting, dizziness, confusion, delirium, stupor, psychosis, coma, and even death, in the worst cases [12,13]. Thus, there is great concern about SA’s presence in both urban and industrial wastewaters, its removal before discharge to the aquatic environment having received a great deal of attention in recent years [14,15,16,17]. As a matter of fact, among 140 CECs, SA was recently appointed as one of the 18 that should be regulated for wastewater discharge [18].
Several studies have demonstrated the potential of phycoremediation for the removal of PPCPs from water [19,20,21,22]. Microalgae are unicellular or multicellular photosynthetic microorganisms that constitute the oldest life forms found in aquatic habitats, in which they are the primary producers of the food chain. Their use for water treatment encompasses several benefits related to carbon dioxide fixation [23,24], nutrients removal [25] and the generation of biomass that is susceptible to energy valorization [26,27] and to obtain algae-derived valuable products [23,28,29]. Furthermore, microalgae have antioxidative mechanisms with enzymes such as catalase and superoxide dismutase that protect them against toxicity and are able to develop physiological and molecular strategies that enable them to tolerate and survive these toxic effects [21]. Microalgal removal of PPCPs mainly occurs by three different mechanisms, namely biosorption, bioaccumulation, and biodegradation, which may combine with each other [21]. The removal of SA by different microalgae strains has been assessed in several studies [14,25,30,31,32,33,34]. According to the recent literature review by Tolboom et al. [19], in comparison with other pharmaceuticals, the removal of SA by microalgae-based treatments is quite high and biodegradation is the main removal mechanism [19].
Biodegradation is the catalytic disintegration of organic compounds into different metabolic intermediates, or, upon conclusion, to carbon dioxide and water. It is probably the most efficient process for the removal of PPCPs [22]. The biodegradation of PPCPs by microalgae can occur through two different routes: (i) metabolic degradation, in which the organic contaminant is the only carbon source or electron donor/acceptor; and (ii) co-metabolism, in which other substrates act as electron donors. Furthermore, biodegradation by microalgae may involve extracellular and intracellular processes, or a combination of both (e.g., extracellular biodegradation occurs initially and then the breakdown products are further degraded intracellularly). A main issue regarding biodegradation is that metabolic intermediates resulting from this process may prevail long after the parent compounds and involve equal or greater risks for the environment and/or public health than the parent compounds. Some studies have been carried out to determine the metabolic intermediates (also known as transformation products) from microalgae-based treatments aimed at the removal of different PPCPs, such as antibiotics [35,36] or hormones [37,38,39] but, to the best of our knowledge, there is no information about SA removal. In any case, from a practical perspective on wastewater treatment, the identification of every single metabolic intermediate from the biodegradation of any organic compound is unaffordable and ultimately unnecessary [40].
In the above context, the present work aimed to explore for the very first time whether microalgae can effectively remove SA from water and simultaneously mitigate its associated toxic effects. For such a purpose, three commonly used microalgal strains in water treatment, namely Chlorella vulgaris, Chlorella sorokiniana, and Scenedesmus obliquus, were tested for reducing SA concentration and its toxic effects on embryos of zebrafish (Danio rerio).
The zebrafish model was used since it is a well-established bioassay that has gained prominence as a preferred model organism in toxicology due to its small size, resilience, short lifecycle, high fecundity, and simple laboratory maintenance [41,42,43]. Its transparent embryos enable direct observation of development, making it ideal for small-scale, economical, and high-throughput studies [43]. Furthermore, the genetic similarity of zebrafish to humans has been highlighted and leveraged to explore disturbances such as intellectual disability, autism spectrum disorders, and the mechanisms underlying various mental and physical health conditions [42]. Recent research has reinforced the utilization of zebrafish for examining the toxicity of different PPCPs [41,44,45,46,47,48,49,50], including the UV filter ethylhexyl salicylate [51,52] and SA [53].

2. Materials and Methods

2.1. Removal of Salicylic Acid from Water by Microalgae

2.1.1. Microalgae and Photobioreactors Operation

Three different microalgae strains were tested for the removal of SA from water, namely Chlorella sorokiniana (CCAP 211/8 K, UTEX Culture Collection), Chlorella vulgaris (SAG 221-12, SAG Culture Collection), and Scenedesmus obliquus (SAG 276-1, SAG Culture Collection), referred to from now on as CS, CV and SO, respectively. Bubbling column photobioreactors (PBRs) with a spherical base, 4 cm diameter, 30 cm height and 250 mL operating volume were used to culture microalgae as described elsewhere [25]. For each strain, microalgae were first precultured in the standard culture medium Mann and Myers [54] and then used to inoculate the PBR, where SA (C7H6O3, ≥99%) provided by Panreac (Panreac Química S.L.U., Barcelona, Spain) was spiked at an initial concentration of 25,000 µg L−1.
Operation of PBRs was performed under batch conditions and a controlled constant temperature (25 ± 1 °C), irradiance (370 µE m−2 s−1) and photoperiod (12:12). Irradiation was accomplished using 8 fluorescent lamps (58 W, 2150 lumen, Philips, France). Aeration was carried out by the injection of CO2 (7% v/v)-enriched air at a rate of 0.3 v/v/min to keep a constant pH (pH = 7.5 ± 0.5) as controlled by a pH sensor. Air employed for aeration was previously filtered through a 0.2 µm sterile air-venting filter (Millex-FG50, Millipore).
For each strain, there were three replicates of both experiments (culture medium + SA + microalgae) and negative controls (culture medium + SA). Throughout the operation, 5 mL aliquots were daily sampled from each PBR to determine the biomass and SA concentrations so to monitor microalgae growth and SA removal, respectively. It was verified that SA concentration in negative controls remained stable throughout the treatment, confirming that its reduction in experiments was associated with microalgae.

2.1.2. Analytical Methods and Instrumentation

Withdrawn aliquots were used for the quantification of biomass concentration (BC) and SA concentration. The BC was determined by optical density at 680 nm (OD680) using a UV–visible spectrophotometer (BECKMAN DU640). Equations (1)–(3), which were established on the basis of dry weight determinations, as described by Escapa et al. [25], were, respectively, applied for CS, CV and SO (R2 ≥ 0.99).
OD680 = 5.1834 BC + 0.0128
OD680 = 2.7933 BC + 0.0317
OD680 = 2.0098 BC + 0.0451
Next, after double centrifugation of the aliquots at 5974 × g for 10 min (SIGMA 2-16P centrifuge), the liquid phase was analyzed for SA concentration by a Waters HPLC 600 equipped with a 2487 dual λ absorbance detector at a detection wavelength of 236 nm. A Phenomenex Gemini-NX C18 column (5 μm, 250 mm × 4.6 mm) was used for separation with a mobile phase consisting of a mixture of acetonitrile/ultrapure water/ortophosphoric acid (70:30:0.1, v/v/v). HPLC grade acetonitrile (CH3CN) and orthophosphoric acid (H3PO4) were provided by DBH Prolabo Chemicals, while ultrapure water was obtained by a Millipore System. After preparation, the mobile phase was filtered through a Millipore membrane (0.45 µm pore diameter) and ultrasonicated for degasification (30 min).

2.2. Toxicity Assessment of Salicylic Acid Solutions and Microalgae Treated Effluents by Zebrafish Embryo Bioassays

2.2.1. Zebrafish Embryo Bioassays

Adult wild-type zebrafish were used as breeding stocks under the conditions described by Soares et al. [55] Briefly, adult specimens were kept in 70 L aquaria with freshwater (tap water after dichlorination and aeration in a recirculation system with both mechanical and biological filters) under a temperature of 28 ± 1 °C, a photoperiod of 14:10 h (light–dark) and ad libitum feeding twice a day with commercial fish diet Tetramin (Tetra, Melle, Germany) supplemented with live brine shrimp (Artemia spp.).
In the afternoon before breeding, adult males and females (2:1) were housed in a cage with a glass marbles bottom cover, within a 30 L aquarium with the same type of water, temperature, and photoperiod as above described. Spawning and fertilization of the eggs were stimulated by the beginning of the light period. At 1–1.5 h post fertilization (hpf), the fertilized eggs were collected from the bottom of the aquarium and then repeatedly washed with water to remove detritus and avoid microorganisms’ proliferation during the experiments [56].
Static water renewal toxicological tests were carried out with the zebrafish embryos following the Fish Embryo Acute Toxicity (FET) test (TG 236), with adaptations [57] set by the Organization for Economic Cooperation and Development (OECD). Briefly, collected and washed fertilized eggs were randomly distributed among 24-wells plates. Using a magnifying glass for observation, 10 fertilized eggs were placed in each well previously filled with 2 mL of the corresponding fresh SA solution, solvent control or control. The SA solutions were prepared by dilution of SA in the standard microalgae culture medium Mann and Myers [54] and freshwater (dechlorinated and aerated tap water in a recirculation system with mechanical and biological filters) in a 1:1 (v/v) ratio. Such a dilution was used to avoid effects of the Mann and Myers medium on the zebrafish embryo development, which was defined after a preliminary toxicity assessment on different ratios of the medium to freshwater (namely, 1:7; 1:3; 1:1, and 1:0 v/v). Next, incubation of the 24-well plates was performed at 26.5 °C over 144 h. The medium was retrieved daily to maintain the concentration of SA and/or dissolved oxygen (DO) and also to avoid microorganisms’ proliferation. Upon retrieving the medium, if present, dead embryos and chorions after hatching were removed.
Four observation time-points, as representative development stages of zebrafish embryo, were set: (i) gastrula period (75%-epiboly stage), (ii) pharyngula period (prim 15-16), (iii) larval stage (protruding-mouth), and (iv) juvenile, which were, respectively, observed at 8, 32, 80 and 144 hpf. An inverted microscope (Nikon Eclipse 5100T) equipped with a digital camera (Nikon D5-Fi2) and a microscope camera controller (Nikon’s Digital Sight DS-U3) were used to observe the embryos at the defined time-points. Photographs of embryos taken at each of these stages have been included as Supplementary Material (Figure S1) together with sketches of developmental stages defined by Kimmel et al. [58].
The mortality rate and morphological abnormalities of zebrafish embryos were assessed at each of the abovementioned observation time-points. Furthermore, the 75% epiboly stage at 8 hpf, hatching rate at 80 hpf and larval length at 144 hpf were also evaluated in accordance with the OEDC Guidelines [57]. Morphological abnormalities were considered to be anomalies in the head, eyes, tail or yolk sac, developmental delay, abnormal cells, pericardial oedema, opaque chorion, excess or lack of pigmentation, lateral position, reduced mobility and involuntary movements.

2.2.2. Validation of the Application of Zebrafish Embryo Assays for the Target Assessment

At the first stage, the above-described procedure was carried out to validate the application of the zebrafish embryo bioassays to assess the toxic effects of SA in the range of concentrations of the microalgae-based treatment. To encompass the range of influent and effluent concentrations in the subsequent microalgae water treatments (please, see Section 2.2), solutions of SA with six different concentrations, namely 25,000; 12,500; 6250; 2500; 250; and 25 µg L−1 were prepared and tested as described. Bioassays were carried out in parallel for solutions of SA at each of these concentrations, solvent controls (Mann and Myers medium: freshwater, 1:1) and controls (freshwater), testing six replicates in all cases. It is necessary to highlight that the controls showed mortality rates equal or lower than 10% in all cases, confirming that the bioassays were properly run.

2.2.3. Toxic Effects of Treated Effluents on Zebrafish Embryo

When the microalgae culture reached the exponential growth phase, PBR were stopped and 75 mL of the effluents were collected, homogenized, and centrifuged twice at 5974× g for 10 min (SIGMA 2-16P centrifuge). After centrifugation, the supernatant was separated and diluted with freshwater (dechlorinated and aerated tap water in a recirculation system with mechanical and biological filters) at a dilution of 1:1 (v/v), as previously set. Also, zebrafish embryo bioassays were carried out for another dilution, namely an effluent to freshwater ratio of 1:9 (v/v) to further verify the effects observations. Effluents from the microalgae PBR at the two aforementioned dilution levels were tested for zebrafish embryo toxicity as already described (Section 2.2.2).

2.2.4. Statistics

The statistical analysis of results was carried out with SPPS Statistics software version 21. The Kolmogorov–Smirnov test and Levene test, were, respectively, used to confirm that data were normal and with homogeneous variance. Upon normality absence, data were analyzed by the Krustal–Wallis test, followed by the Mann–Whitney U test for pairwise comparisons with the Bonferroni correction. Significance was defined at p ≤ 0.05. Statistical analyses were carried out for data obtained at 8, 32, 80 and 144 hpf. Solvent and experimental controls were grouped when no significant differences between them were detected. To increase statistical power, all abnormalities detected were grouped to determine the percentage of total abnormalities, as the percentage of embryos with one or more abnormalities in comparison to the control.

3. Results

3.1. Removal of Salicylic Acid from Water by Microalgae

Results on the concentration of SA in aliquots withdrawn from PBR throughout the microalgae-based batch treatment are depicted in Figure 1 together with microalgae biomass concentration. As can be seen, either for CS, CV, or SO, the concentration of SA progressively decreased with time, while microalgae biomass concentration increased.
Regarding the SA concentration reduction during the microalgae-based treatments, it can be seen that the efficiency of the strains considered was quite different. The first strain to attain the steady state was CS, while SO and CV took longer. As for the SA concentration, SO was the strain that provided the largest reduction, with a final concentration of around 1700 µg L−1, followed by CS with 9600 µg L−1 and CV with 18,700 µg L−1. Thus, the three strains were able to reduce SA concentration but to different extents, varying from a reduction larger than 93% by SO, to nearly 40% by CS and just 25% by CV. As for the biomass concentration increase during batch culture, differences between the three microalgae strains were also evident, with SO being the strain that showed the largest growth with a final biomass concentration of 4.3 g L−1, followed by CV (3.0 g L−1) and CS (2.1 g L−1).

3.2. Toxicity Assessment of Salicylic Acid Solutions and Microalgae Treated Effluents by Zebrafish Embryo Bioassays

3.2.1. Validation of the Application of Zebrafish Embryo Assays for the Target Assessment

The results on the mortality and total abnormalities in zebrafish embryos caused by SA solutions are depicted in Figure 2, and the observed abnormalities are detailed in Table 1.
As for the mortality rates (Figure 2a), the observed values were between 4% and 60%. The mortality rates corresponding to SA solutions with concentrations of 25,000 and 12,500 µg L−1 were significantly higher than the control rate from the first to the last observation time-point (8 hpf to 144 hpf, respectively). In the case of the 6250 and 2500 µg L−1 SA solutions, the associated mortality rates were significantly higher than for the control from 32 hpf and 80 hpf, respectively, to 144 hpf. For the SA solutions with 250 and 25 µg L−1 of SA, the observed mortality rates were not significantly different from controls at any observation time-point. Regarding total abnormalities (Figure 2b), significantly higher values for the control were observed for the three SA solutions with the largest concentrations (25,000, 12,500 and 6250 µg L−1) from the first observation time-point (8 hpf). Then, for the subsequent observation time-points, the total abnormalities were significant for all the tested SA concentrations, except for 25 µg L−1, which resulted in a significantly higher value than the control at 32 hpf due to the observation of some cases of developmental delay that were not verified at later observation time-points (Table 1).
As can be seen in Table 1, at the first observation time-point (8 hpf), the total abnormalities identified in Figure 2 for the three highest SA concentrations, namely 25,000, 12,500 and 6250 µg L−1, were related both to the 75% epiboly rate and developmental delay. Then, at 32 hpf, zebrafish embryo developmental delay was verified to be significant when exposed to SA solutions with any of the considered concentrations, but the larger the concentration, the larger the percentage of observations within 1.7 and 100%. Such a growing trend with SA concentration was verified for the lack of pigmentation at 32 hpf (40.2 to 100%) for embryos exposed to SA concentrations from 2500 to 25,00 µg L−1 (p ≤ 0.05). Also, opaque chorion was significant at 32 hpf for embryos exposed to 25,000 and 12,500 µg L−1 of SA. At 80 hpf, an excess of pigmentation was observed on larvae exposed to concentrations ≥ 250 µg L−1 (p ≤ 0.05) within 4.5 and 96.7%; the larger the concentration, the larger the percentage. These percentages remained at 144 hpf, when, moreover, embryos in a lateral position were observed for those specimens exposed to SA concentrations ≥ 2,500 µg L−1 (p ≤ 0.05). Apart from results depicted in Table 1, it was also verified that SA did not affect the hatching rate at any of the tested concentrations, and 100% of embryos hatched in all cases. Moreover, the results on the larval length at 144 hpf for embryos exposed to SA solutions were not significantly different from the controls.
The results in Figure 2 and Table 1 show a clear increasing trend of observed effects (%) on zebrafish embryos with SA concentration, especially the mortality rate. There is also a certain increasing trend with time for the mortality rate, with the maximum values being attained at 80 hpf and then maintaining at 144 hpf (Figure 2). In the case of total abnormalities, such a trend is observable from 8 to 32 hpf (Figure 2), then remaining similar values at longer observation time-points.

3.2.2. Toxic Effects of Treated Effluents on Zebrafish Embryo

Figure 3 illustrates the results of the mortality and total abnormalities observed in zebrafish embryos exposed to the effluents from the microalgae treatment of SA-contaminated water, while the details on the observed abnormalities are depicted in Table 2. Additionally, the observed effects upon exposure to diluted treated effluents are displayed in Table S1 as Supplementary Material. Pictures of zebrafish embryos and some of the observed abnormalities are showed in Figure 4.
The mortality rates in Figure 3a evidence differences between effluents from the treatments by the different strains. In the case of CV, the mortality rates were significantly higher than those observed for the corresponding controls from the first (8 hpf) to the last (144 hpf) observation time-points. For effluents from CS treatment, the mortality rates were significantly higher than those for controls at the last observation time-points, namely 80 and 144 hpf. It was only for effluents from the SO treatment that no significant mortality rates were observed in zebrafish embryos at any observation time-point.
Embryos exposed to CV-treated effluents showed total abnormalities (%) that, as was the case for mortality rates, were significantly higher than those observed for the corresponding controls from the first (8 hpf) to the last (144 hpf) observation time-points. Then, from the 32 hpf to 144 hpf, the percentage of total abnormalities was also significantly elevated for the effluents from CS. Differently, in the case of effluents from the SO treatment, the total abnormalities were significant only at 32 hpf, but not at later observation times. Indeed, for the three strain-treated effluents, the highest values of total abnormalities were observed at 32 hpf. Overall, it can be seen that the higher the percentage of SA removal by the microalgae strain (Figure 1, SO > CS > CV), the smaller the total abnormalities (%) caused by exposure to the corresponding effluent (Figure 3b, SO < CS < CV).
Regarding anomalies observed in zebrafish embryos after exposure to microalgae-treated effluents, Table 2 shows that effects were observed for CV-treated water from the first observation time-point (8 hpf). This effluent caused a significant diminution of the 75%-epiboly rate and a significant developmental delay in comparison to the control. Then, at 32 hpf, the developmental delay was significant for embryos exposed to the effluents treated by the three strains here considered, with the observations decreasing in the order CV > CS > SO, which is the same order as the final concentrations from each treatment in Figure 1. In the case of effluents from the SO treatment, no abnormalities except developmental delay were observed at 32 hpf, although such a delay ceased at longer observation times (Table 2) and it was not observed for embryos exposed to the 1:9 dilution of this effluent (Table S1, within Supplementary Material). Differently, apart from developmental delay, at 32 hpf, effluents from CS and especially from CV also caused a significant lack of pigmentation in the exposed embryos. At a later hpf, exposure to these effluents also resulted in a significant excess of pigmentation at 80 hpf. Such excess of pigmentation of embryos exposed to effluents from CS and CV was still significant at 144 hpf, with embryos exposed to CV effluent also showing a significant occurrence of a lateral position. It was evident that the exposure of zebrafish embryos to the effluents from CV treatment resulted in significant effects at all the observation time-points with the largest (the lowest in the case of 75%-epiboly rate) observed incidences in this work. According to these results, it is clear that the larger the remaining SA concentration at the end of microalgae treatment, the larger the incidence of toxic effects in exposed zebrafish embryos. This was further confirmed by the results obtained for embryos exposed to the 1:9 dilution, as can be seen in Table S1, within the Supplementary Material, which evidences that the more efficient the microalgae removal of SA from water, the smaller the incidence of effects on zebrafish embryos.

4. Discussion

The microalgae strains used in this work, namely CS, CV, and SO, were able to remove SA from water, and, in parallel, their biomass increased during cultivation in PBR. However, differences among them were quite evident. The strain SO was the most efficient in the removal of SA, as illustrated in Figure 1, and was also the strain for which biomass experienced the most remarkable growth. These differences corroborate findings in preceding research work.
In a previous study, SO was also shown to be more efficient than CS and CV in the removal of another NSAID, namely diclofenac [59]. Likewise, the superiority of SO in the removal of diclofenac compared with other strains was also confirmed by Sánchez-Sandoval et al. [60]. In the removal of other pharmaceuticals, SO (also known as Tetradesmus obliquus) has been confirmed to be especially capable when compared with other microalgae strains [61,62], which has been related to the fact that SO is quite resistant and prone to accumulating lipophilic pharmaceuticals [62,63]. Regardless, CS has been shown to be more efficient than SO in the removal of acetaminophen, which is a highly consumed over-the-counter analgesic and antipyretic drug [49]. As for CV, although comparatively less efficient than SO and CS in the removal of SA (this study), diclofenac [59] and acetaminophen [49], it has been reported to efficiently remove other pharmaceuticals such as clomipramine, trihexyphenidyl, flecainide, orphenadrine or memantine, mainly by biodegradative mechanisms [62]. Thus, the efficiency of removing different pharmaceuticals is different depending on the species and the strain. In fact, the effectiveness of microalgal systems in wastewater treatment strongly relies on biotic factors such as the selected microalgae species and inoculum concentration [64]. However, abiotic factors such as pH, hydraulic retention time (HRT), light intensity and CO2/O2 supply also have a crucial role in the removal of pollutants from water [64]. In the specific case of SA, the influence of both biotic and abiotic factors is evident by the different efficiencies reported in the literature, as shown in Table S3. As can be seen, the range goes from negative values (associated with the increase in SA concentration due to biotransformation of different compounds or release from the solid phase) to 99% removal. As for the mechanisms, although microalgae removal of pharmaceuticals may occur by bioaccumulation and bioadsorption, biodegradation is the main mode [22,65,66], which has been verified for SA [19]. Biodegradation involves a series of complex catalytic reactions that mostly result in the transformation of pharmaceuticals into different metabolic intermediates (rarely, complete degradation, namely until CO2 and water, is attained) [22]. These metabolic intermediates may be toxic, thus compromising the efficiency of microalgae removal. Therefore, zebrafish embryo bioassays were carried out in this work to evaluate effluents’ toxicity.
First, zebrafish embryo bioassays were validated for the assessment of SA in the range of concentrations used in this study. Within such a range, the mortality of zebrafish (Figure 2a) was verified at SA concentrations equal to or higher than 2500 µg L−1. This relatively high mortality associated with SA is in line with the results obtained by Conde-Valcells et al. [67], who carried out the same procedure (OECD test guideline 236 [57]). These authors observed a 100% lethality after 3 days of exposure to SA at 10−3 M (≈138,120 µg L−1) and determined that the SA concentration causing 50% lethality (LC50) at 72 hpf was 3.8 10−5 M (≈5248 µg L−1) and that the lowest effect level (LEL) for mortality was 1.0 10−5 M (≈1381 µg L−1). Higher LC50 were observed by Ali et al. [53] who, following a different procedure, determined values of 47,500, 47,500, and 46,700 µg L−1 at 24, 48 and 72 hpf, respectively. It is important to highlight that, even compared with other NSAIDs, there are very few published studies on SA toxicity [68]. Compared to diclofenac, the effects on zebrafish mortality caused by SA were first observed at 8 hpf versus 32 hpf for diclofenac, but lower mortality percentages were determined at relatively long exposure times, with 100% mortality having been observed for dicloflenac concentrations equal to or higher than 6250 µg L−1 at 144 hpf [59]. Regarding zebrafish embryo abnormalities under exposure to SA (Figure 2b), these were observed as soon as 8 hpf for concentrations equal to or higher than 6250 µg L−1. For subsequent observation time-points, namely 32, 80 and 144 hpf, abnormalities were observed for embryos subjected to SA concentrations equal to or higher than 250 µg L−1, with the incidence of occurrence increasing with observation time and SA concentration. The observed abnormalities (Table 1) were a decrease in the 75%-epiboly rate (8 hpf), development delay (8 to 32 pf), opaque chorion (32 hpf), pigmentation issues (32 to 144 hpf) and lateral position (144 hpf). Published results on the abnormalities caused by SA exposure on zebrafish are scarce. At 5 days post-fertilization, Ali et al. [69] observed a significant incidence of yolk sac oedema in zebrafish exposed to 30,000 µg L−1 SA. Using an SA concentration of 400 µM (≈55,248 µg L−1), Liu et al. [70] observed a significant increase in the incidence of abnormalities (severe deformities in the neurocranium and the viscerocranium) at 96 hpf.
After confirming the reduction in SA concentration along microalgae treatment in the operated PBR and validating the application of zebrafish bioassays for the assessment of SA toxicity within the concentration range of interest in this work, bioassays were carried out to assess effluents’ toxicity. Given that a dilution ratio of 1:1 of effluents to freshwater was used to minimize the effects of the culture medium on the embryos, the toxicity results obtained for diluted effluents (Figure 3, Table 2) are to be compared to those obtained for the 12,500 µg L−1 of SA in Figure 2 and Table 1, which corresponds to the dilution of the feeding concentration (25,000 µg L−1). Such a comparison showed that the three strains here used were not only able to remove SA, as evidenced in Figure 1, but also to provide a reduction in zebrafish embryo mortality and abnormalities incidence. However, as it may be seen in Figure 3, the efficiency of these strains in the reduction in toxic effects was quite different. At the last observation time-point (144 hpf), mortality and abnormalities incidence caused by exposure to the SA solution of 12,500 µg L−1 were 41% and 94%, respectively (Figure 2). As can be seen in Table 2, such a SA concentration caused excess of pigmentation and lateral position effects in 94% and 3% of the embryos, respectively. Comparatively, the effluent from the treatment with SO, which was the most efficient in the reduction in SA concentration, caused an incidence (at 144 hpf) of 12% and 2% of mortality and abnormalities, respectively. However, these values were not significantly different from those determined for the control, so it can be said that the SO treatment practically removed the SA toxicity completely. In the case of CS, which was second most efficient in the removal of SA, the mortality and abnormalities incidence at 144 hpf were, respectively, 18% and 6% (significantly higher than control’s values). Finally, CV was the strain that provided the smallest reduction in SA concentration (Figure 1) and, correspondingly, the toxicity effects of the corresponding effluent were visibly larger than for SO and CS effluents. In fact, mortality incidence was not significantly different from that determined for the SA solution of 12,500 µg L−1, while the percentage of abnormalities was reduced to 63%. Therefore, not only the removal of SA, but also the decrease in associated toxic effects is strain-dependent. In fact, a correspondence between SA concentration in treated water by these strains and the associated toxic effects was observed. For example, SA concentration in the effluent from CS treatment was ≈6000 µg L−1 and, considering the dilution (1:1), mortality and abnormalities incidences resembled those determined for the solution of 2500 µg L−1 (19% and 5%, respectively). Thus, it may be inferred that biodegradation of SA by these strains does not involve the generation of metabolic intermediates with larger toxic effects on zebrafish embryos than SA.
It is well known that microalgae-based treatments for the removal of pharmaceuticals from water may result in the generation of toxic metabolic intermediates since biodegradation is the main removal mechanism [22,65,66,71]. Recently, results have been reported on the determination of metabolic intermediates from the microalgae degradation of this sort of pollutant [38,72,73,74,75,76]. Despite the importance of these results for inferring degradation routes, from a safety perspective of water treatment, it is necessary to find out if it provides a significant decrease in toxicity [77]. The toxicity of effluents from different wastewater treatments has been assessed by different authors to complement pharmaceutical(s) removal results, with zebrafish being the most commonly used model [77,78,79,80,81]. To the best of our knowledge, in the case of microalgae-based treatments, such assessment has been carried out just for diclofenac [59] and acetaminophen [49] in previous studies using the same strains considered here. In comparison with the reduction in diclofenac concentration (67% to 99%, with SO being the most efficient, while CS and CV provided similar removals) [59], and acetaminophen concentration (17% to 67%, with the efficiency of strains being CS > SO > CV) [49], the removal of SA determined in this work was intermediate (25 to 93%, with the efficiency of strains being SO > CS > CV). These results highlight that efficiency for each pharmaceutical is highly strain-dependent. For the three pharmaceuticals, a correspondence between effluent concentration and toxicity effects was verified, with the greater the microalgae efficiency in reducing pharmaceutical concentration, the greater the efficiency in reducing toxicity effects. García-Camberro et al. [77] recently proved that toxicity evaluation using zebrafish embryos was even more sensitive than physical–chemical monitoring of tertiary effluents from wastewater treatment plants, providing a very sensitive tool to ascertain wastewater suitability. In this work, zebrafish was confirmed to be, together with concentration monitoring, an appropriate model to assess the performance of microalgae in the removal of pharmaceuticals.

5. Conclusions

Microalgae-based treatment was implemented in this work to remove SA from water, the efficiency being assessed in terms of the reduction in both SA concentration and toxic effects on zebrafish embryos. The three different microalgae strains tested here, namely CV, CS, and SO, showed distinct capacities in SA uptake. Average concentration reductions of 25, 40, and 93% from the feeding concentration (25,000 µg L−1), were respectively attained by each strain at the end of the treatment. Zebrafish embryo bioassays were validated in the SA concentration working range in this work (25 to 25,000 µg L−1), evidencing mortality effects at concentrations equal to or higher than 2500 µg L−1 and the increase in abnormalities incidence at concentrations equal to or higher than 250 µg L−1. It was then shown that the three considered microalgae strains provided a decrease in toxicity effects on zebrafish embryos throughout treatment. However, their efficiency was distinct, namely SO > CS > CV, resembling the reduction in SA concentration. The obtained results pointed out large specific differences between microalgae strains in their capacity to remove SA from water and the importance of carrying out toxicity complementary assessments for the evaluation of effluents safety, with zebrafish embryos being an adequate model for such a purpose.

Supplementary Materials

The following supporting information can be downloaded at: https://mdpi.longhoe.net/article/10.3390/w16131874/s1, Figure S1: Images of zebrafish at different developmental stages: (a,b) gastrula stage; (c,d) pharyngula stage; (e,f) larval stage. Note: Sketches are from Kimmel et al. (1995) while pictures were taken from the microscope. Table S1: Effects on zebrafish embryo exposed to effluents (diluted 1:9 with freshwater) from the microalgae-based treatment of salicylic acid contaminated water. Note: Mean results (n= 12 for control; n = 6 exposed groups) are shown together with standard errors. Results significantly different from control (p ≤ 0.05) are highlighted in bold. Table S2: Published results on the removal of salicylic acid (SA) from different aqueous matrices by microalgae-based treatment.

Author Contributions

Conceptualization, M.O. and M.M.S.; experimental design, M.M.S., C.E. and M.O.; methodology, M.M.S., C.E. and M.O.; experimentation, C.E. and R.N.C.; supervision, M.M.S. and M.O.; data analysis and discussion, C.E., R.N.C., M.A.K., M.O., T.N. and M.M.S.; writing—original draft preparation, C.E., R.N.C., M.A.K. and M.O.; writing—review and editing, M.M.S., T.N. and M.O. All authors have read and agreed to the published version of the manuscript.

Funding

This research received funding by Fundação para a Ciência e a Tecnologia (FCT, Lisboa, Portugal), grant number IF/00314/2015. Carla Escapa was supported by the Ministerio de Educación, Cultura y Deportes (Madrid, Spain), which awarded her with a grant for a stay (EST15/00405) at the Interdisciplinary Centre of Marine and Environmental Research (CIIMAR).

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

Acknowledgments

Moonis Ali Khan acknowledges financial support through the Researchers Supporting Project (RSP2024R345), King Saud University, Riyadh, Saudi Arabia.

Conflicts of Interest

The authors declare no conflicts of interest and that funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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Water 16 01874 g001
Figure 1. Concentration of salicylic acid (SA) throughout the culture of three different microalgae strains, namely Chlorella sorokiniana (CS), Chlorella vulgaris (CV) and Scenedesmus obliquus (SO) is represented in the primary axis (filled symbols). For each strain, microalgae biomass determined at each cultivation time is represented in the secondary axis (hollow symbols. Note: Average results are depicted together with error bars standing for standard deviations (n = 3).
Figure 1. Concentration of salicylic acid (SA) throughout the culture of three different microalgae strains, namely Chlorella sorokiniana (CS), Chlorella vulgaris (CV) and Scenedesmus obliquus (SO) is represented in the primary axis (filled symbols). For each strain, microalgae biomass determined at each cultivation time is represented in the secondary axis (hollow symbols. Note: Average results are depicted together with error bars standing for standard deviations (n = 3).
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Figure 2. Mortality (a) and total abnormalities (b) observed in zebrafish embryos exposed to salicylic acid (SA) solutions with different concentrations (µg L−1), as specified in the legend, at each observation time-point (8, 32, 80 and 144 h post fertilization (hpf)). Note: Average results are depicted together with error bars standing for standard deviations (n = 12 for controls; n = 6 exposed groups). Asterisks (*) identify those results that were significantly higher than the control (p ≤ 0.05).
Figure 2. Mortality (a) and total abnormalities (b) observed in zebrafish embryos exposed to salicylic acid (SA) solutions with different concentrations (µg L−1), as specified in the legend, at each observation time-point (8, 32, 80 and 144 h post fertilization (hpf)). Note: Average results are depicted together with error bars standing for standard deviations (n = 12 for controls; n = 6 exposed groups). Asterisks (*) identify those results that were significantly higher than the control (p ≤ 0.05).
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Figure 3. Mortality (a) and total abnormalities (b) observed in zebrafish embryos exposed to effluents from microalgae treatment of salicylic acid (SA)-contaminated water. Results are given for effluents from treatment by Chlorella sorokiniana (CS), Chlorella vulgaris (CS) and Scenedesmus obliquus (SO) at each observation time-point (8, 32, 80 and 144 h post fertilization (hpf)). Note: Average results are depicted together with error bars standing for standard deviations (n = 12 for controls; n = 6 exposed groups). Asterisks (*) identify those results that were significantly higher from the control (p ≤ 0.05).
Figure 3. Mortality (a) and total abnormalities (b) observed in zebrafish embryos exposed to effluents from microalgae treatment of salicylic acid (SA)-contaminated water. Results are given for effluents from treatment by Chlorella sorokiniana (CS), Chlorella vulgaris (CS) and Scenedesmus obliquus (SO) at each observation time-point (8, 32, 80 and 144 h post fertilization (hpf)). Note: Average results are depicted together with error bars standing for standard deviations (n = 12 for controls; n = 6 exposed groups). Asterisks (*) identify those results that were significantly higher from the control (p ≤ 0.05).
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Figure 4. Images of zebrafish embryo at different observation time-points (8, 32, 80 and 144 h post fertilization (hpf)). At 8 hpf: (a) developmental delay; at 32 hpf: (b) normal development, (c) developmental delay, (d) opaque corium; at 80 hpf: (e) lateral position and abnormality (yolk-sac oedema); at 144 hpf: (f) normal development, (g) lateral position, (h) excess of pigmentation; (i) abnormality.
Figure 4. Images of zebrafish embryo at different observation time-points (8, 32, 80 and 144 h post fertilization (hpf)). At 8 hpf: (a) developmental delay; at 32 hpf: (b) normal development, (c) developmental delay, (d) opaque corium; at 80 hpf: (e) lateral position and abnormality (yolk-sac oedema); at 144 hpf: (f) normal development, (g) lateral position, (h) excess of pigmentation; (i) abnormality.
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Table 1. Abnormalities observed in zebrafish embryos exposed to salicylic acid (SA) solutions with different concentrations (µg L−1) at each observation time-point (8, 32, 80 and 144 h post fertilization (hpf)). Note: Average results are depicted together with standard deviations (n = 12 for controls; n = 6 exposed groups). Results significantly higher than the control (p ≤ 0.05) are identified in bold.
Table 1. Abnormalities observed in zebrafish embryos exposed to salicylic acid (SA) solutions with different concentrations (µg L−1) at each observation time-point (8, 32, 80 and 144 h post fertilization (hpf)). Note: Average results are depicted together with standard deviations (n = 12 for controls; n = 6 exposed groups). Results significantly higher than the control (p ≤ 0.05) are identified in bold.
Observation Time-PointSA Concentration (µg L−1)75%-Epiboly
Rate		Developmental
Delay		Opaque
Chorion		Lack of
Pigmentation		Excess of
pigmentation		Lateral
Position
8 hpf0 (control)92.4 ± 7.74.6 ± 5.3
25,00058.3 ± 9.831.5 ± 9.5
12,50062.1 ± 10.324.5 ± 6.6
625075.0 ± 5.514.4 ± 5.2
250090.1 ± 6.01.7 ± 4.0
25094.2 ± 5.10.8 ± 2.9
2598.3 ± 4.11.7 ± 4.1
32 hpf0 (control) 0.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.0
25,000 100 ± 0.055.2 ± 12.1100 ± 0.00.0 ± 0.0
12,500 91.7 ± 10.238.4 ± 8.291.7 ± 10.20.0 ± 0.0
6250 68.8 ± 8.70.0 ± 0.068.8 ± 8.70.0 ± 0.0
2500 40.2 ± 10.70.0 ± 0.040.2 ± 10.70.0 ± 0.0
250 5.3 ± 5.50.0 ± 0.00.0 ± 0.00.0 ± 0.0
25 1.7 ± 4.10.0 ± 0.00.0 ± 0.00.0 ± 0.0
80 hpf0 (control) 0.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.0
25,000 0.0 ± 0.00.0 ± 0.00.0 ± 0.096.7 ± 8.20.0 ± 0.0
12,500 0.0 ± 0.00.0 ± 0.00.0 ± 0.094.4 ± 8.60.0 ± 0.0
6250 0.0 ± 0.00.0 ± 0.00.0 ± 0.060.4 ± 9.70.0 ± 0.0
2500 0.0 ± 0.00.0 ± 0.00.0 ± 0.05.0 ± 6.20.0 ± 0.0
250 0.0 ± 0.00.0 ± 0.00.0 ± 0.04.5 ± 5.60.0 ± 0.0
25 0.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.0
144 hpf0 (control) 0.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.00.7 ± 2.8
25,000 0.0 ± 0.00.0 ± 0.00.0 ± 0.096.7 ± 8.225.8 ± 7.4
12,500 0.0 ± 0.00.0 ± 0.00.0 ± 0.094.4 ± 8.62.8 ± 6.8
6250 0.0 ± 0.00.0 ± 0.00.0 ± 0.060.4 ± 9.72.4 ± 5.8
2500 0.0 ± 0.00.0 ± 0.00.0 ± 0.05.0 ± 6.25.0 ± 6.2
250 0.0 ± 0.00.0 ± 0.00.0 ± 0.04.6 ± 5.70.0 ± 0.0
25 0.0 ± 0.00.0 ± 0.00.0 ± 0.01.7 ± 4.10.0 ± 0.0
Table 2. Abnormalities observed in zebrafish embryos exposed to effluents from microalgae treatment of salicylic acid (SA)-contaminated water. Results are given for effluents from treatment by Chlorella sorokiniana (CS), Chlorella vulgaris (CS) and Scenedesmus obliquus (SO) at each observation time-point (8, 32, 80 and 144 h post fertilization (hpf)). Note: Average results are depicted together with standard deviations (n = 12 for controls; n = 6 exposed groups). Results significantly different from the control (p ≤ 0.05) are identified in bold.
Table 2. Abnormalities observed in zebrafish embryos exposed to effluents from microalgae treatment of salicylic acid (SA)-contaminated water. Results are given for effluents from treatment by Chlorella sorokiniana (CS), Chlorella vulgaris (CS) and Scenedesmus obliquus (SO) at each observation time-point (8, 32, 80 and 144 h post fertilization (hpf)). Note: Average results are depicted together with standard deviations (n = 12 for controls; n = 6 exposed groups). Results significantly different from the control (p ≤ 0.05) are identified in bold.
Observation
Time-Point		SA Concentration
(µg L−1)		75%-Epiboly
Rate		Developmental
Delay		Opaque ChorionLack of
Pigmentation		Excess of
Pigmentation		Lateral
Position
8 hpfControl95.8 ± 5.11.7 ± 3.9
CS93.1 ± 5.30.0 ± 0.0
CV68.3 ± 7.517.7 ± 6.5
SO93.3 ± 8.25.0 ± 8.4
32 hpfControl 1.8 ± 4.10.0 ± 0.00.0 ± 0.00.0 ± 0.0
CS 37.9 ± 12.20.0 ± 0.037.9 ± 12.20.0 ± 0.0
CV 90.7 ± 14.80.0 ± 0.090.7 ± 14.80.0 ± 0.0
SO 12.8 ± 4.70.0 ± 0.00.0 ± 0.00.0 ± 0.0
80 hpfControl 0.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.0
CS 0.0 ± 0.00.0 ± 0.00.0 ± 0.06.1 ± 6.90.0 ± 0.0
CV 0.0 ± 0.00.0 ± 0.00.0 ± 0.063.3 ± 3.70.0 ± 0.0
SO 0.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.0
144 hpfControl 0.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.0
CS 0.0 ± 0.00.0 ± 0.00.0 ± 0.06.4 ± 7.20.0 ± 0.0
CV 0.0 ± 0.00.0 ± 0.00.0 ± 0.063.3 ± 3.76.7 ± 10.3
SO 0.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.0
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Escapa, C.; Coimbra, R.N.; Khan, M.A.; Neuparth, T.; Santos, M.M.; Otero, M. Assessing the Efficiency of Microalgae in the Removal of Salicylic Acid from Contaminated Water: Insights from Zebrafish Embryo Toxicity Tests. Water 2024, 16, 1874. https://doi.org/10.3390/w16131874

AMA Style

Escapa C, Coimbra RN, Khan MA, Neuparth T, Santos MM, Otero M. Assessing the Efficiency of Microalgae in the Removal of Salicylic Acid from Contaminated Water: Insights from Zebrafish Embryo Toxicity Tests. Water. 2024; 16(13):1874. https://doi.org/10.3390/w16131874

Chicago/Turabian Style

Escapa, Carla, Ricardo N. Coimbra, Moonis Ali Khan, Teresa Neuparth, Miguel Machado Santos, and Marta Otero. 2024. "Assessing the Efficiency of Microalgae in the Removal of Salicylic Acid from Contaminated Water: Insights from Zebrafish Embryo Toxicity Tests" Water 16, no. 13: 1874. https://doi.org/10.3390/w16131874

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