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Article

Ecotoxicological Assessment of Perfluorooctane Sulfonate and Perfluorooctanoic Acid Following Biodegradation: Insights from Daphnia magna Toxicity and Yeast Estrogen Screen Assays

by
Muyasu Grace Kibambe
* and
Maggy Ndombo Benteke Momba
Department of Environmental, Water and Earth Sciences, Tshwane University of Technology, Arcadia Campus, Private Bag X680, Pretoria 0001, South Africa
*
Author to whom correspondence should be addressed.
Water 2025, 17(23), 3368; https://doi.org/10.3390/w17233368
Submission received: 27 September 2025 / Revised: 3 November 2025 / Accepted: 5 November 2025 / Published: 26 November 2025
(This article belongs to the Section Water Quality and Contamination)
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Versions Notes

Abstract

Perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) pose significant health risks through various exposure pathways, including ingestion of contaminated food and water, as well as dermal absorption. Aquatic organisms are especially at risk, as water bodies serve as primary pathways for the transport and transformation of these chemicals. While the biodegradation study was previously performed using a bacterial consortium from the activated sludge compartment at Zeekoegat WWTP, the ecotoxicological implications of the treated effluents remained unclear, particularly given the potential presence of degradation products. To address this gap, the present study used bioassays to evaluate the acute toxicity and endocrine-disrupting potential of PFOS and PFOA. For this purpose, PFOS and PFOA concentrations ranged from 58 ng/L to 1050 ng/L, and two types of bioassays were used: the Daphnia magna acute toxicity test, which examined the short-term lethal effects of the samples on a small freshwater organism (Daphnia magna), and the Yeast Estrogen Screen (YES), which measured estrogenic activity, an important indicator of potential endocrine disruption. Results revealed detectable estrogenic activity at environmentally relevant concentrations, with PFOS showing higher activity than PFOA. The estradiol equivalency (EEQ) values in samples containing PFOA ranged from 0.23 ± 0.029 ng/L to 3.15 ± 0.056 ng/L and from 0.43 ± 0.036 ng/L to 1.96 ± 0.086 ng/L in samples containing PFOS. Daphnia magna bioassays showed 100% mortality in samples containing PFOS at concentrations ≥ 62 ng/L and in samples containing PFOA at concentrations ≥ 142 ng/L, classifying them as ‘Very High Acute Hazard’ falling into Hazard Class V (100% mortality) according to the classification system proposed in 2003 by Persoone and co-workers. These bioassays helped to determine whether the degradation products were more toxic compared to the parent compounds, thereby supporting the objective of this study to assess environmental safety post-treatment.

1. Introduction

Persistent chemicals from domestic and industrial activities have become widespread in the environment due to their long half-lives, recalcitrant nature, and bioaccumulative properties [1]. Among these, perfluoroalkyl substances (PFASs) are increasingly detected in soil, sediment, and water at concentrations ranging from picograms to nanograms per liter [2]. These chemicals are usually used as lubricants, corrosion inhibitors, and wetting agents in stain-resistant treatments for leather, paper, and clothing, and in foam fire extinguishers due to their ability to repel both water and oil and their special chemical and thermal stability [3,4]. As a result, growing global concern has been raised over the rising levels of these emerging contaminants in the environment. Per- and polyfluoroalkyl substances are released into the environment through manufacturing, product use, distribution, and disposal, with wastewater treatment plant (WWTP) effluents serving as one of the primary pathways into aquatic systems [5,6].
Among the per- and polyfluoroalkyl substances, perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are the most detected in environmental matrices. Due to their hydrophobic nature, they tend to adsorb onto suspended particles and sediments [7,8,9]. Perfluorooctanoic acid and perfluorooctane sulfonate continue to enter the aquatic systems, and a significant proportion of the global population will continually be exposed to these chemicals [10,11]. These chemicals are known to be harmful to freshwater organisms such as algae, fish, and Daphnia magna [10,12]. Furthermore, they have been linked to plastic pollution and endocrine disruption [13]. Perfluorooctane sulfonate (PFOS) and PFOA exhibit varying degrees of toxicity in aquatic organisms, with PFOS generally showing stronger effects in acute exposure studies [2,14].
In South Africa, PFAS concentrations have also been reported in wastewater treatment plants at environmentally relevant levels. According to PFOS and PFOA concentrations in influent and effluent samples from the Daspoort, Phola, and Zeekoegat WWTPs ranged from approximately 9 to 146 ng/L for PFOS and 2 to 15 ng/L for PFOA, with Daspoort showing the highest concentrations compared to the other sites. These findings confirm that the concentration range (58–1050 ng/L) used in the present study reflects values observed in local wastewater treatment systems and thus represents realistic environmental exposure levels.
Additionally, the International Agency for Research on Cancer (IARC) has classified PFOA as “possibly carcinogenic to humans” [11]. Epidemiological studies have shown a strong association between exposure to PFOA and PFOS and an increased risk of testicular and kidney cancer [15]. Although these chemicals are widely detected in surface waters and wildlife, their toxic effects on aquatic ecosystems are still not fully understood [16]. As PFOS and PFOA concentrations continue to rise in aquatic environments, the potential for toxicity and ecological risks to aquatic organisms increases as well [17].
The effluent samples used in this study were previously used in the investigation of biodegradation of PFOS and PFOA using a bacterial consortium derived from the activated sludge compartment at the Zeekoegat WWTP. Partial degradation of both compounds over a 45-day incubation period was observed. However, the ecotoxicological implications of the treated effluents remained unclear, particularly regarding potential transformation products. The present study, therefore, extends this earlier work by evaluating the acute toxicity and endocrine-disrupting potential of PFOS and PFOA after biodegradation, using Daphnia magna and Yeast Estrogen Screen (YES) bioassays.
Although many studies have focused primarily on the chemical removal of PFAS [18,19], few have examined the residual toxicity and estrogenic activity of the resulting effluents. Assessing the ecological safety of these effluents is therefore critical in determining whether they can be safely discharged into the environment or require additional treatment.
Given the persistence and bioaccumulative nature of PFAS in aquatic environments, sensitive bioassays are essential to assess their ecotoxicological impacts. A variety of assays are available for this purpose, broadly classified into in vivo and in vitro approaches [20,21]. Daphnia species, among the oldest and most widely used model organisms in aquatic toxicology, serve as primary bioindicators of water quality in freshwater systems [22]. Standardized acute and chronic assays with Daphnia magna provide critical insights into the effects of pollutants, including PFASs [23]. Complementing in vivo testing, the recombinant Yeast Estrogen Screen (YES) assay has gained wide acceptance as an in vitro tool for detecting estrogenic and anti-estrogenic activity due to its simplicity, reliability, and suitability for environmental monitoring [24,25,26]. This assay uses a genetically modified Saccharomyces cerevisiae strain expressing the human estrogen receptor α and a lacZ reporter gene, enabling quantification of estrogenic activity through β-galactosidase induction and colorimetric change [27,28].
In this study, two tests were used to assess the ecotoxicological and endocrine-disrupting effects of PFOS and PFOA after biodegradation. By combining in vivo (Daphnia magna) assays with in vitro (YES assay) analysis, the study provides a comprehensive evaluation of PFAS toxicity. This combined approach highlights the importance of complementary bioassays in understanding the ecological risks posed by persistent pollutants such as PFOS and PFOA in freshwater ecosystems.

2. Materials and Methods

2.1. Chemicals

Analytical-grade PFOS and PFOA were purchased from Wellington Laboratories (Guelph, ON, Canada). All reagents used for the preparation of the minimum medium (M63) [29] were purchased from Sigma Aldrich (Aston Manor, South Africa). All chemicals were used as received without further purification. Primary stock solutions of PFOA and PFOS were prepared in methanol (MeOH) at a concentration of 1 mg/L and stored at 4 °C in amber glassware to prevent light degradation. Working standard mixtures were prepared by appropriately diluting the stock with M63 minimal medium to initiate the biodegradation experiments. Therefore, the effluents from the biodegradation experiments used in the ecotoxicity and estrogenicity assays contained PFAS in M63 medium, with methanol levels diluted to well below 0.1%.

2.2. Biodegradation Process

The bacterial consortium from Zeekoegat WWTP was used to assess the biodegradation potential of PFOA and PFOS in 250 mL Erlenmeyer flasks over 45 days. Initial inoculum densities were optimized using the Box–Behnken Design and averaged to 1.31OD600 (≈3 × 108 CFU/mL). Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) concentrations ranging from 150 to 1050 ng/L were prepared from a 1 mg/L methanol stock solution and added to M63 minimal medium (pH adjusted to neutral with 1 M HCl or NaOH). Erlenmeyer flasks were incubated at 30 °C, 100 rpm, for 45 days in the dark to prevent photodegradation. Sterile control flasks (no inoculum) were also included. Aliquots (10 mL) were sampled every five days for LC–MS/MS analysis of PFOA and PFOS degradation. To ensure representative sampling, flasks were vortexed before each withdrawal, and aliquots were homogenized prior to analysis.
The effluents from the biodegradation experiments were used for subsequent toxicity and estrogenic activity assays (the Daphnia magna acute toxicity test and the Yeast Estrogen Screen assay). Although PFOA and PFOS commercial standards were originally supplied in methanol, working solutions were prepared and added to the M63 minimal medium for the biodegradation experiments. Consequently, all ecotoxicity assays were performed using biodegradation samples in M63 medium without direct methanol spiking, and the methanol concentration was negligible and below the 0.1% threshold.

2.3. Chemical Analysis

Analysis and quantification of PFOA and PFOS were performed using a Shimadzu LC-MS-8030 system (Shimadzu USA Manufacturing Inc., Canby, OR, USA) equipped with an electrospray ionization (ESI) source operating in negative ion mode. Multiple reaction monitoring (MRM) transitions were optimized via flow injection analysis, with precursor/product ions, collision energies, and cone voltages provided in Table S1. Chromatographic separation was achieved on a C18 polar column using 20 mM ammonium acetate in water and methanol as the mobile phase. Quantification was based on external multi-point calibration curves (R2 > 0.99) and validated with quality control samples and blanks. The method detection (LOD) and quantification limits (LOQ) were determined at 3× and 10× signal-to-noise ratios, respectively, with values summarized in Table S2.
All glassware was rinsed with methanol and oven-dried to prevent contamination. Samples and blanks were spiked with a surrogate standard (MPFHxS18O2, 100 ng/mL), and recovery tests yielded 74–90% with relative standard deviations below 10%. The method was confirmed to be repeatable, sensitive, and linear across all measurements.

2.4. The Recombinant Yeast Estrogen Screen (YES)

2.4.1. Sample Preparation

Extraction of the biodegradation effluent medium for estrogenic activity testing was performed in triplicate according to the method described by Aneck-Hahn et al. [30]. Briefly, the pH of the effluent was adjusted to 3 using hydrochloric acid. Solid-phase extraction (SPE) cartridges were conditioned sequentially with 5 mL of double-distilled water (ddH2O), 5 mL of methanol (MeOH), and another 5 mL of ddH2O before 20 mL of the sample was loaded. The cartridges were dried under vacuum for 20 min to remove residual solvents. Analytes were eluted with 6 mL of MeOH, and the eluates were evaporated to near dryness under a gentle stream of nitrogen. Triplicate samples were then delivered to the Environmental Chemical Pollution and Health Research Laboratory at the University of Pretoria, where all dried extracts were reconstituted in 2 mL of HPLC-grade ethanol. The extracts were transferred to amber glass vials and stored at −20 °C until analysis.

2.4.2. The Recombinant Yeast Estrogen Assay

The yeast strain was obtained from Xenometrix, Allschwil, Switzerland (Cat. No. N05-230-E). Stock cultures and growth medium were prepared, and the YES assay was performed according to the procedure previously described by De Jager et al. [31]. The yeast growth medium (5 mL) was inoculated with 50 µL of the 10× concentrated yeast stock and incubated at 28 °C in a rotating water bath at 150–155 rpm until turbid (approximately 24 h). Serial dilutions were made of the extracts and controls in 96-well microtiter plates (untreated, clear, flat-bottom) in HPLC-grade ethanol. From the dilution plate, 10 µL aliquots were then transferred to 96-well assay plates and allowed to evaporate to dryness. Aliquots (200 µL) of the assay medium containing the yeast and chromogenic substrate (CPRG) were then dispensed into each sample well. Each YES assay plate contained at least one row of blanks (assay medium with solvent ethanol) and a full standard curve for 17β-estradiol (E2; Cat. No. E8875, Sigma-Aldrich, St. Louis, MO, USA), ranging from 1 × 10−8 M to 1.2 × 10−15 M (2.7 × 10−6 to 3.2 × 10−13 g/L). Solvent controls were used to determine the detection limit for each plate (mean blank + 3 × SD), and sample EEQs were interpolated from the corresponding E2 standard curve.
The plates were sealed with autoclave tape and placed in a naturally ventilated incubator at 29 °C for 3 to 5 days. After 3 days of incubation, the color development of the medium was checked for 3 days (day 3 to 5) at an absorbance (abs) of 540 nm for color change and 620 nm for turbidity of the yeast culture. The absorbance was measured on a Multiskan Spectrum 96-well plate reader (Thermo Fisher Scientific, Waltham, MA, USA) to obtain data with the best contrast. All experiments were performed in triplicate. The following equation was applied to correct for turbidity:
Corrected value = test abs (540 nm) − [test abs (620 nm) − median blank abs (620 nm)]
The E2 standard curve was fitted (sigmoidal function, variable slope) using Graphpad Prism (version 4), which calculated the minimum, maximum, slope, EC50 value, and 95% confidence limits. The detection limit (dl) of the yeast assay was calculated as absorbance elicited by the solvent control (blank) plus three times the standard deviation. The limit of quantification (loq) was equivalent to the EC10 of the E2 standard curve. Cytotoxicity was indicated if the absorbance of a cell was below the absorbance elicited by the solvent control (blank) minus three times the standard deviation. Estradiol equivalents (EEQ) of samples above the Limit of Quantification (LOQ) were interpolated from the estradiol standard curve and corrected with the appropriate dilution factor for each sample. Figure S1 shows the standard dose–response curve for 17β-estradiol (E2) used in the YES assay. The curve was fitted with a sigmoidal function using GraphPad Prism, and EEQ values for test samples were interpolated from this curve.
The YES assay was performed on biodegradation samples collected at day 0 (initial PFAS concentrations of 150, 300, 450, 600, 750, 900, and 1050 ng/L) and at day 45 (residual PFAS concentrations), representing untreated influents and treated effluents, respectively, to evaluate whether estrogenic activity decreased after biodegradation.

2.5. Daphnia magna Acute Toxicity Test (Water Flea)

A 48 h acute toxicity screening assay was conducted using Daphnia magna, following standard test procedures described by USEPA [32] and acceptability criteria outlined by Slabbert [33]. The purpose of the assay was to evaluate the potential toxicity of test samples by observing the survival response of the test organisms. The Daphnia magna was less than 48 h old and represented the grazer trophic level. Tests were performed under static-renewal conditions using moderately hard reconstituted water as the control and dilution medium. The reconstituted water refers to the OECD M4 medium prepared according to OECD Guideline 202 [34]. This medium was used for both culturing and testing Daphnia magna and consisted of defined mineral salts (CaCl2·2H2O, MgSO4·7H2O, NaHCO3, and KCl) to simulate natural freshwater conditions. Each test was conducted in 4 replicate chambers, with 5 organisms per chamber (20 organisms per test sample in total), as shown in Table 1. Negative controls (reconstituted water only) were included, and control mortality remained ≤10%, fulfilling OECD guideline criteria.
Physicochemical parameters (pH, temperature, dissolved oxygen, and electrical conductivity [EC]) were measured before and after the bioassay in accordance with Standard Methods [35]. During the bioassay, these parameters were monitored to ensure that water quality conditions remained within acceptable ranges for Daphnia magna survival, as outlined in OECD Guidelines 202 and 211 [34,36]. The purpose of the test was not to determine the intrinsic toxicity of pure PFOS or PFOA but rather to evaluate the overall ecotoxicity of the effluents collected after the biodegradation process, which contained residual PFAS and potential transformation products.
Daphnia magna acute toxicity assays were performed on undiluted (100% v/v) biodegradation effluents collected on day 45 at different initial PFAS concentrations (150–1050 ng/L). No additional spiking or solvent was added; thus, the test concentrations reflected the levels of PFOA, PFOS, and any byproducts that remained after biodegradation.
Daphnia magna used in the tests were cultured under standard laboratory conditions prior to exposure, and all toxicity assays, including the control, were conducted using effluents derived from M64 minimal medium to ensure comparable matrix composition. Toxicity was assessed based on immobilization (including mortality) of Daphnia magna according to the USEPA [32] method for effluent toxicity testing, with reference to OECD Guideline 202 [34] for immobilization criteria and test validity, rather than as EC50 values. The study focused on assessing the safety of the effluents after biodegradation rather than the toxicity of pure PFAS.

2.6. Hazard Classification for Natural Waters

The ecological risk or hazard class of each water sample was determined using the DEEEP (Direct Estimation of Ecological Effect Potential) protocol [33], in accordance with the hazard classification system for natural waters proposed by Persoone et al. [37]. This classification reflects the level of acute or chronic ecological risk associated with the sample. For each bioassay in the screening battery, a percentage effect (PE) was calculated based on the observed response, such as immobility or mortality in animal tests, or inhibition in microbial or algal assays. Based on these PE values, the sample was categorized into one of five hazard classes (Table 2), using either screening or definitive test criteria.

2.7. Statistical Analysis

Statistical significance was evaluated using single-factor analysis of variance (ANOVA) in Microsoft Excel 2020 (Office 365) with the Real Statistics add-in. The analysis was used to compare EEQ values among different treatment groups in the YES assay. Statistical significance was established at p < 0.05.

3. Results

3.1. Results of the Recombinant Yeast Estrogen Assay

Figure 1 shows the colorimetric results of the YES bioassay after a 3-day incubation period. Positive wells (rows G and H) exhibited a deep red color accompanied by yeast growth, while the negative control wells remained light orange (row F). These results confirm that some test samples retained estrogenic activity even after the biodegradation process, highlighting the importance of both chemical and biological assessment methods when evaluating PFAS-treated effluents.
The estrogenic activity before and after biodegradation was assessed using the Yeast Estrogen Screen (YES) assay to evaluate any changes in estrogenic activity at various concentrations of PFOA and PFOS, as summarized in Table 3 and Table 4, respectively. It is worth mentioning that only influent and effluent samples from the biodegradation experiment were analyzed to evaluate the reduction in estradiol equivalent (EEQ) concentrations. In samples containing PFOA, the EEQ values ranged from 0.72 ± 0.065 to 3.15 ± 0.056 ng/L in the influent and from 0.23 ± 0.029 ng/L to 0.99 ± 0.108 ng/L in the effluent. In samples containing PFOS, the EEQ values ranged from 0.43 ± 0.036 ng/L to 1.96 ± 0.086 ng/L in the influent and from 0.64 ± 0.039 ng/L to 1.16 ± 0.079 ng/L in the effluent. The effluent generally showed lower EEQ values compared to the influent in both samples containing PFOA and PFOS, indicating a reduction in estrogenic activity.
However, an increase in EEQ was observed in samples containing 300 ng/L PFOA, where the EEQ values increased from 0.72 ± 0.065 ng/L to 0.99 ± 0.108 ng/L. Similarly, in samples containing 300 ng/L and 600 ng/L PFOS, the EEQ values increased from 0.84 ± 0.065 ng/L to 1.16 ± 0.079 ng/L and from 0.43 ± 0.036 ng/L to 0.89 ± 0.041 ng/L, respectively. Cytotoxicity was only observed in samples containing 900 ng/L, likely due to nonspecific toxic effects at higher PFAS concentrations that inhibited yeast growth and β-galactosidase activity in the YES assay, Table 4.
Figure 2 compares PFOA and PFOS in terms of how much estrogenic activity was reduced from influent to effluent. In samples containing PFOA, the highest removal efficiency of 81% was observed at an initial concentration of 450 ng/L, while the lowest removal efficiency of 10% was observed in samples containing 750 ng/L. For samples containing PFOS, the highest removal efficiency of 46% occurred at an initial concentration of 900 ng/L, whereas the lowest removal of 2% was observed in samples containing 750 ng/L. No removal was observed in the sample containing an initial concentration of 300 ng/L for both PFOA and PFOS, as well as in the sample with an initial concentration of 600 ng/L for PFOS.

3.2. Daphnia magna Screening Assays

Table 5 and Table 6 summarize the results of Daphnia magna exposed to various concentrations of PFOA and PFOS after 24 and 48 h of exposure. The acute toxicity assays were conducted on effluents obtained after 45 days of incubation, since in real-world conditions treated effluents are ultimately discharged into aquatic environments. Daphnia magna, a standard freshwater bioindicator species, was used to evaluate whether the treated water remained toxic. The effective concentration for 50% (EC50) values was not calculated because the assays were designed to assess effluent safety rather than to establish benchmark toxicity thresholds for pure compounds. Instead, mortality (%) at each concentration was reported to provide direct evidence of the ecotoxicological risk posed by the effluents.
The test samples were considered valid, as no mortality (≤10%) was observed in the controls for both PFOA and PFOS assays. A 100% mortality rate was observed at both 24 and 48 h in the undiluted samples, which is classified as a very high acute hazard (Class V) according to the Hazard Classification System for Screening Tests (Persoone et al. [37]), except in samples containing 58 ng/L and 72 ng/L PFOA, where 50% mortality was recorded, indicating acute hazard (Class III) (Table 5). In contrast, 100% mortality was observed in all samples containing PFOS at both 24 and 48 h across the tested concentrations (62–811 ng/L). According to the Hazard Classification System for Screening Tests [37], this response is classified as a very high acute hazard (Class V). Unlike PFOA, where some effluents showed 50% mortality at lower concentrations (58 and 72 ng/L, Class III), PFOS effluents consistently exhibited complete lethality, indicating higher acute toxicity potential.
Electrical conductivity (EC), pH, dissolved oxygen (DO), and temperature were measured at the start and end of the bioassay using a handheld Hach HQ40D multiparameter meter to monitor water quality. In samples containing PFOA, EC ranged from 4.25 to 6.06 mS/cm, with a much lower EC of approximately 0.3 mS/cm observed in the control. The pH values varied between 6.05 and 7.53, DO ranged from 3.22 to 5.21 mg/L, and temperature remained within 16.5 °C to 21.2 °C. In samples containing PFOS, EC ranged from 5.02 to 6.46 mS/cm, pH from 5.76 to 7.22, DO from 3.71 to 5.21 mg/L, and temperature from 19.0 °C to 20.4 °C. Physical parameters monitored in the study are summarized in Tables S3 and S4.
Statistical analysis revealed that, for samples containing PFOA, only DO had a significant influence (p < 0.05) on the estrogenic activity (EEQ values), whereas pH, temperature, and EC did not exhibit any significant effect (p > 0.05). Similarly, in PFOS-containing samples, none of the parameters (pH, temperature, DO, or EC) showed any statistically significant effect (p > 0.05) on the EEQ values.
Overall, temperature during the bioassay remained within the optimal range for Daphnia magna (16.5–21.2 °C), while pH ranged between 5.76 and 7.53. EC increased slightly with higher PFAS concentrations, particularly for PFOS (up to 6.46 mS/cm), and DO levels consistently exceeded 3.0 mg/L. These stable physicochemical conditions confirm that the observed toxicity responses were attributable to PFAS exposure rather than fluctuations in water quality.

4. Discussion

4.1. Reduction in Estrogenic Activity During the YES Bioassay

Unlike conventional YES assays designed to derive median effective concentration (EC50) values from serial dilutions of pure chemicals, this study applied the assay to compare influent (before treatment) and effluent (after treatment) samples. The YES bioassay was used to quantify estradiol equivalent concentrations (EEQs) in samples containing varying concentrations of PFOA and PFOS, both before and after biodegradation. This approach allowed direct assessment of biodegradation efficiency in reducing estrogenic activity and provided insight into whether the treated effluents still posed endocrine-disrupting risks or could be considered environmentally safe.
The highest estrogenic activity for PFOA was observed in the effluent of the sample containing 72 ng/L, with an EEQ value of 0.99 ± 0.108 ng/L. Similarly, for PFOS, the highest estrogenic activity was recorded in the effluent of the sample containing 139 ng/L, with an EEQ value of 1.16 ± 0.079 ng/L. According to Genthe and Steyn [38], the trigger value for estrogenic activity in drinking water is 0.7 ng/L, suggesting potential concern for human exposure. In the present study, only PFOA samples with effluent concentrations of 142 ng/L, 241 ng/L, and 596 ng/L exhibited EEQ values below this threshold, specifically 0.23 ± 0.029 ng/L, 0.51 ± 0.063 ng/L, and 0.66 ± 0.027 ng/L, respectively (Table 3). In comparison, among the PFOS samples, only the 148 ng/L concentration resulted in an EEQ value below the trigger level, at 0.64 ± 0.039 ng/L (Table 4). Although the trigger value is established for drinking water, it is relevant in this context because effluent from the biodegradation process is ultimately discharged into surface waters, rivers, or groundwater systems, where it may affect downstream water quality and potentially impact drinking water sources.
A reduction in EEQ values was observed in almost all effluent samples, indicating that the biodegradation process effectively reduced estrogenic activity. This decrease is environmentally significant, as reduced estrogenicity in effluents suggests a lower potential risk to aquatic organisms and downstream water resources. For example, in samples with an initial PFOA concentration of 450 ng/L, EEQ decreased from 1.21 ± 0.070 ng/L to 0.23 ± 0.029 ng/L after biodegradation, indicating successful reduction in estrogenicity. However, in the sample with an initial concentration of 300 ng/L PFOA, EEQ values increased from 0.72 ± 0.065 ng/L to 0.99 ± 0.108 ng/L. Although this value remained below the critical threshold of 1 ng/L, it approached levels at which estrogenic effects may occur in fish, indicating that, although partial biodegradation occurs, the effluents may still pose ecotoxicological risks to aquatic organisms [39,40]. These results also revealed that there was no significant difference (p > 0.05) between the influent and effluent EEQ values in samples containing PFOA.
The EEQ thresholds are usually applied to steroidal estrogens such as estrone (E1), estradiol (E2), and estriol (E3). However, recent studies have shown that PFOS and PFOA may enhance estrogenic responses or exert weak estrogenic activity in vitro [41,42]. Therefore, the detection of EEQs in this study suggests that even partially degraded PFAS residues may contribute to endocrine disruption, especially when considering the presence of other contaminants in a complex wastewater matrix. Furthermore, no cytotoxicity was detected in any sample, suggesting that the observed estrogenic activity at the reported EEQ concentrations did not harm the yeast cells used in the assay (Table 3). In samples containing PFOS, EEQ values also decreased from influent to effluent, as can be seen in Table 4. Despite this observation, it is important to note that samples with an initial concentration of 300 ng/L and 600 ng/L depicted an increase in the EEQ values in the effluent, indicating a rise in estrogenicity after treatment. This could be due to the biodegradation by-products produced during partial degradation that retain or enhance the estrogen receptor (ER)-binding affinity or the presence of metabolites with greater estrogenic activity from microbial transformation of parent compounds [43,44]. These results also revealed that there was no significant difference (p > 0.05) between the influent and effluent EEQ values in samples containing PFOS. A cytotoxicity effect was reported in samples containing a high initial concentration of 900 ng/L; this effect can be due to the accumulation of toxic degradation intermediates and the high concentration interfering with cell viability. The cytotoxicity observed in this study aligns with existing literature indicating that PFAS can induce cytotoxic effects, particularly with increasing exposure time and concentration. For instance, Wee and Aris [11] observed cytotoxic responses in human cell lines exposed to PFOS, suggesting interference with membrane integrity and mitochondrial function at elevated concentrations. Wielsøe et al. [45] reported EC50 values for PFOS and PFOA in HepG2 liver cells ranging from 20 to 200 µM after 24 h exposure, indicating concentration-dependent cytotoxicity. Furthermore, Kjeldsen and Bonefeld-Jørgensen [41] indicated that PFAS can act as endocrine disruptors and may impair cell viability by interacting with intracellular targets, particularly at high concentrations. Florentin et al. [46] showed that both PFOA and PFOS had a cytotoxic effect on human HepG2 cells but did not cause DNA damage nor micronuclei formation in tested cells. Additionally, the Organization for Economic Co-operation and Development (OECD) and the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) have acknowledged that longer exposure durations (up to 96 h) improve the detection of cytotoxic responses [47]. Thus, the observed cytotoxicity in our sample may be a result of both the high PFOS concentration and the extended exposure time, which is consistent with the time-dependent cytotoxic nature of PFAS reported in the literature.
The endocrine-disrupting properties of PFAS compounds have been widely studied in ecological systems, but direct EEQ values for PFOS and PFOA are rarely reported. However, several studies have assessed their estrogenic potential using in vitro assays [48,49]. Kjeldsen and Bonefeld-Jørgensen [41] reported that PFOS and PFOA exhibited weak or indirect estrogenic effects, primarily through potentiation of 17β-estradiol in breast cancer cell lines and estrogen receptor (ER) reporter gene assays. These findings align with our findings, where YES assay responses suggest low but detectable estrogenic activity in certain contaminated samples with PFOA and PFOS. The estrogenic responses observed in the present study may be attributed to the ability of PFOA and PFOS to interact directly with the estrogen receptor (ER), particularly within the ligand-binding domain (LBD) [50]. These interactions can induce receptor-mediated transcription, which the YES assay detects as β-galactosidase activity. Furthermore, the variation in EEQ values across samples may reflect the influence of the intracellular environment on PFAS behavior, including possible transformation into metabolites with altered estrogenic potential. Although a reduction in PFOA and PFOS concentrations was observed from the influent to the effluent after biodegradation, the EEQ values did not reduce proportionally, suggesting that biodegradation does not eliminate estrogenic potential in a sample.
The percentage removal in the present study ranged from 2% to 81%, as reported in Figure 2. Overall, samples containing PFOA showed a higher EEQ percentage removal efficiency compared to PFOS. The highest EEQ removal (81%) was achieved at an initial concentration of 450 ng/L, whereas the EEQ removal was lower (34%) at the same concentration. Another observation was that, in samples containing an initial concentration of 1050 ng/L, a high removal efficiency of 75% was recorded for PFOA, while only 12% removal was observed for PFOS. These findings suggest that biodegradation was more effective in reducing estrogenic activity in samples containing PFOA compared to those containing PFOS. In samples containing a lower concentration of 150 ng/L, both PFOA and PFOS exhibited moderate EEQ removal; however, PFOA showed a higher removal efficiency (55%) compared to PFOS (32%). Interestingly, at 750 ng/L, both compounds showed very low EEQ removal (Figure 2). The lower EEQ removal efficiency observed in the present study, particularly at 750 ng/L, could be attributed to microbial inhibition caused by PFAS toxicity. The highest EEQ removal efficiencies observed in samples containing PFOA as compared to PFOS are in alignment with a study from Huang and Jaffé [51], which highlights that certain microbial communities can initiate defluorination of PFOA more readily than PFOS under both aerobic and anaerobic conditions. Furthermore, PFOS exhibited lower EEQ percentage removal in all the samples, suggesting that it is more persistent and resistant to biodegradation processes compared to PFOA. This is consistent with the fact that PFOS is known to be more chemically stable and bioaccumulative than PFOA [52,53,54]. Additionally, only the sample containing an initial concentration of 900 ng/L PFOS exhibited a higher EEQ removal compared to PFOA. This may suggest an adaptive microbial response or could be attributed to experimental variability (Figure 2). Although the EEQ values reported in this study were relatively low, previous research in the Buffalo River in South Africa has demonstrated that environmental concentrations as low as 2.7–13.6 ng/L of estrogenic compounds can induce vitellogenin production in fish, a key biomarker of estrogenic disruption [55]. While PFOA and PFOS are not classical estrogens, the estrogenic activity detected via the YES assay suggests a potential risk, particularly given the persistence and bioaccumulation potential of PFAS compounds in aquatic environments. The estrogenic activity observed in the present study may be attributed to the ability of PFOA and PFOS to interact directly with the estrogen receptor (ER), particularly within the ligand-binding domain (LBD) [56,57]. In some samples, an increase in EEQ was observed after biodegradation, while in others, a reduction was recorded (Table 3 and Table 4). These variations could be explained by the formation of degradation by-products or by changes in environmental conditions, which may have influenced the behavior of PFOS and PFOA and their potential to bind to the ER. This gives rise to concerns that treated effluents may still pose endocrine risks to aquatic ecosystems, particularly if PFOS and PFOA biodegradation is incomplete or results in the formation of estrogenically active metabolites. Notably, these findings align with the main goal of wastewater treatment, which is not only to reduce the concentration of contaminants but also to mitigate their associated biological effects, such as endocrine disruption.

4.2. Acute Toxicity Assessment Using Daphnia magna

Testing biodegradation effluents directly with Daphnia magna reflects real-world exposure scenarios, where treated wastewater is released into aquatic ecosystems. This assay was applied only to effluents, unlike the YES assay, which was conducted on both influent and effluent samples to evaluate the removal efficiency of estrogenic activity. The Daphnia magna test was therefore used to determine whether the treated water retained acute toxicity and to provide practical insight into the ecological safety of biodegradation as a treatment option for PFAS-contaminated water.
Daphnia magna, a freshwater water flea, is a keystone invertebrate crustacean widely used in ecotoxicological testing due to its high sensitivity to pollutants and its ecological importance. As a primary food source for many fish species, Daphnia magna plays a vital role in freshwater trophic dynamics. Notably, approximately 41.24% of all known fish species inhabit freshwater ecosystems [58], highlighting the ecological relevance of assessing chemical impacts at this level of the food web. Therefore, evaluating the toxicity of PFAS compounds such as PFOS and PFOA in Daphnia magna is critical for understanding the potential risks to freshwater biodiversity and ecosystem stability. In this study, an acute toxicity test using Daphnia magna following a biodegradation experiment was performed to assess the residual toxicity of PFOA and PFOS. No mortality was observed in the control samples at both 24 and 48 h, indicating no acute toxicity under control conditions (Table 5 and Table 6). In samples containing PFOA, a mortality rate of 50% was observed at lower concentrations (58 and 72 ng/L) after 48 h, classifying these treatments as posing an acute hazard according to the toxicity hazard potential scale reported by Persoone et al. [37]. An increase in toxicity was observed at higher concentrations ranging from 142 ng/L to 702 ng/L, where 100% mortality was recorded within 48 h, categorizing these treatments as very high acute hazards (Table 5, [37]). In samples containing PFOS, 100% mortality was recorded in all the samples at concentrations ranging from 62 ng/L to 811 ng/L, categorizing these treatments as very high acute hazard [37], except in the controls, where no mortality was observed [Table 6]. This is consistent with findings by Park et al. [59], who observed Class IV and V toxicity in PFAS-contaminated wastewater post-treatment, reinforcing the notion that conventional or biological treatments may not sufficiently mitigate ecological risks. Subsequent studies used this classification system to evaluate the hazard potential of complex waste materials, highlighting the importance of using validated tools to avoid arbitrary hazard classification [60]. In contrast, Wang et al. [8] reported a significant reduction in both estrogenicity and toxicity following complete mineralization of PFOS using advanced oxidation processes, suggesting that the persistence of toxicity in our samples may be due to the formation of partially degraded intermediates during biodegradation. In 2015, a study by Koçbaş and Oral [61] assessed the acute toxicity of municipal wastewaters in Turkey using Daphnia magna as a bioindicator and applied a hazard classification system by converting LC50 values into Toxic Units (TU). This system grouped wastewater into five hazard categories, ranging from Class I (no acute toxicity) to Class V (very high acute toxicity), providing a standardized framework for ecological risk assessment. Using the hazard classification by Persoone et al. [37], the effluent samples in our study fall into Class V, indicating very high acute hazard. This is consistent with findings by Park et al. [59], who observed Class IV and V toxicity in PFAS-contaminated wastewater post-treatment, emphasizing that conventional or biological treatments may not sufficiently mitigate ecological risks. This study directly assessed biodegradation effluents, unlike many previous studies, and demonstrated that even after treatment, water contaminated with PFAS compounds such as PFOA and PFOS may still pose a very high acute hazard to aquatic life.
Among the aquatic invertebrates, Daphnia magna is a widely recognized model organism for evaluating aquatic toxicity. In the present study, Daphnia magna exhibited high mortality rates following exposure to PFOA and PFOS, indicating acute toxicity. These findings are consistent with previous research by Zhang et al. [62], which demonstrated that high PFOS exposure significantly inhibits glutathione S-transferase (GST) activity and impairs the antioxidant defense system in Daphnia magna. Both short- and long-term exposures to PFOS were shown to disrupt physiological functions, suggesting that the observed mortality in our study may result from oxidative stress and compromised detoxification pathways. Previous studies have consistently reported that PFOS is more toxic than PFOA, although many of these investigations were limited to acute toxicity tests or used PFAS concentrations well above environmentally relevant levels (>6 mg/L) [63] In the present study, PFOS demonstrated greater toxicity to Daphnia magna than PFOA, as indicated by reduced survival rates and heightened stress responses. These findings are consistent with earlier research showing that PFOS can be up to ten times more toxic than PFOA in aquatic organisms, though the extent of toxicity varies depending on the species and whether acute or chronic effects are being evaluated [64,65]. Furthermore, PFAS exposure to Daphnia has been linked to decreased reproduction and growth, along with increased mortality, underscoring the sensitivity of this species to PFAS pollution [63,66]. In contrast, the present study assessed toxicity at nanogram-per-liter (ng/L) concentrations, aligning more closely with levels commonly detected in natural water bodies. While acute toxicity may not be apparent at environmentally relevant concentrations, many authors reported that PFOA and PFOS can exert chronic sublethal effects on aquatic organisms. These effects include endocrine disruption, genotoxicity, and oxidative stress [67,68]. The findings of this study highlight the importance of assessing not only the removal efficiency of PFAS compounds during wastewater treatment but also the potential presence of toxic degradation by-products that may persist in the effluent and pose ecological risks. Therefore, beyond evaluating acute toxicity, our results highlight the need to consider long-term sublethal effects associated with PFAS exposure, particularly given their high persistence, chemical stability, and bioaccumulative potential in aquatic environments [69,70].
The results of this study support growing evidence that PFAS compounds, particularly PFOS and PFOA, pose significant ecotoxicological risks to aquatic invertebrates even at sublethal concentrations. Perfluorooctane sulfonate (PFOS) was found to be notably more toxic than PFOA to Daphnia species, aligning with previous studies reporting LC50 values of 63 mg/L for PFOS and 181 mg/L for PFOA [14]. While chronic exposure to PFOS at 50 mg/L has been linked to 50% mortality in Daphnia magna [71], the authors reported adverse survival effects at concentrations as low as 1.0 mg/L in Daphnia carinata. Similarly, reproductive toxicity, such as reduced hatching success, was reported at even lower concentrations [72], emphasizing the vulnerability of lower trophic organisms. Sublethal PFAS exposure has also been associated with endocrine disruption, metabolic alteration, and genotoxicity [67,73], often following non-monotonic dose–response patterns where low-dose effects cannot be predicted from high-dose data [74]. These findings underline the limitations of traditional toxicity assessment approaches and highlight the need for more comprehensive evaluations of PFAS impacts on environmentally relevant concentrations. Our results contribute to existing research by showing that long-term exposure to low levels of PFAS may cause lasting harm to aquatic ecosystems. Future studies should investigate whether testing Daphnia toxicity at lower concentrations through dilution could reveal reduced toxicity.
Consistent physicochemical conditions (pH, EC, DO, and temperature) during the bioassay confirmed that the observed Daphnia magna toxicity was attributable to PFAS exposure rather than variations in water quality.

5. Conclusions

Toxicity and ecotoxicity were assessed in this study following biodegradation experiments. The Yeast Estrogen Screen (YES) assay provided insight into the estrogenic potential of samples containing PFOA and PFOS before and after biodegradation, while acute toxicity in Daphnia magna was evaluated only after treatment. The EEQ values in PFOA samples ranged from 0.23 ± 0.029 to 3.15 ± 0.056 ng/L, while PFOS samples ranged from 0.43 ± 0.036 to 1.96 ± 0.086 ng/L, confirming the potential for endocrine disruption at environmentally relevant concentrations. Acute toxicity tests revealed 100% mortality at PFOS concentrations ≥ 62 ng/L and at PFOA concentrations ≥ 142 ng/L, indicating a significant threat to aquatic ecosystems. Despite reductions in EEQ, most undiluted effluent samples still exhibited very high acute toxicity (Hazard Class V; Persoone et al. [37]). This highlights a critical gap: while biodegradation lowers estrogenic activity, treated effluents may continue to pose acute ecological risks. These findings demonstrate the importance of applying complementary biological assays to capture both chronic and acute effects, as reliance on a single endpoint could underestimate ecological risks.
These findings suggest that wastewater management strategies should go beyond chemical removal alone and also consider residual biological activity and acute toxicity, incorporating bioassay-based monitoring into routine PFAS management to ensure effluent safety before release into the environment. Such measures are essential for protecting the aquatic ecosystems and downstream water quality, including drinking water sources.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/w17233368/s1, Table S1: MRM parameters for the targeted PFAS compounds; Table S2: LOD and LOQ of the targeted PFAS compounds; Table S3: Physical parameters measured at the start and end of the bioassays in sample containing PFOA; Table S4: Physical parameters measured at the start and end of the bioassays in sample containing PFOS; Figure S1: Standard calibration curve of 17β-estradiol (E2) used in the Yeast Estrogen Screen (YES) assay; The LC-MS/MS chromatograms of PFOA and PFOS.

Author Contributions

Conceptualization: M.N.B.M.; data curation: M.G.K.; formal analysis: M.G.K.; funding acquisition: M.N.B.M.; investigation: M.G.K.; methodology: M.G.K.; project administration: M.N.B.M.; resources: M.N.B.M.; supervision: M.N.B.M.; validation: M.G.K.; visualization: M.G.K.; writing—original draft: M.G.K.; review and editing: M.G.K. and M.N.B.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Research Foundation (NRF), South Africa (UID 87310) and the Tshwane University of Technology (TUT) (UID 120188). The Article Processing Charge (APC) was funded by the Department of Environmental, Water and Earth Sciences, Faculty of Science, Tshwane University of Technology, Pretoria, South Africa.

Data Availability Statement

The original contributions presented in this study are included in the article and Supplementary Material. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

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Water 17 03368 g001
Figure 1. Example of a YES plate after 3 days of incubation. Rows A–E contain serial dilutions of the samples, row F represents the vehicle control, row G the positive control, and row H the extended standard curve. Wells in row G show strong red/violet coloration, indicating high estrogenic activity, while row H appears light orange, reflecting lower activity levels.
Figure 1. Example of a YES plate after 3 days of incubation. Rows A–E contain serial dilutions of the samples, row F represents the vehicle control, row G the positive control, and row H the extended standard curve. Wells in row G show strong red/violet coloration, indicating high estrogenic activity, while row H appears light orange, reflecting lower activity levels.
Water 17 03368 g001
Water 17 03368 g002
Figure 2. Comparison of PFOA and PFOS in terms of the influent-to-effluent removal efficiency of estrogenicity.
Figure 2. Comparison of PFOA and PFOS in terms of the influent-to-effluent removal efficiency of estrogenicity.
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Table 1. Summary of test conditions and test acceptability criteria for Daphnia magna acute toxicity tests with effluents and receiving waters [33].
Table 1. Summary of test conditions and test acceptability criteria for Daphnia magna acute toxicity tests with effluents and receiving waters [33].
Summary of Toxicity Test
Test systemDaphnia test
Test speciesDaphnia magna
Age of test organismsLess than 48 h old
Trophic levelAcute toxicity
Test procedureUSEPA, 2002 [32]
Summary of test conditions for Daphnia magna acute toxicity test
Test typeStatic renewal
Water temperature20 °C ± 1 °C; or 25 °C ± 1 °C
Light qualitySAmbient laboratory illumination
Photoperiod8 h dark: 16 h light
Feeding regimeFeed algae and commercial fish flakes while in holding prior to the test
AerationNone
Size of test chamber50 mL
Volume of test sample25 mL
Number of test organisms per chamber5
Number of replicate chambers4
Total number of test organisms per sample20
Control and dilution waterModerately hard, reconstituted water
Test duration48 h
Effect measuredPercentage lethality (no movement on gentle prodding) calculated in relation to control
Test acceptability90% or greater survival in control
InterpretationLethality > 10% indicates toxicity if control lethality is ≤10%
Table 2. Hazard classification system for screening tests [37].
Table 2. Hazard classification system for screening tests [37].
ClassDescription
CLASS INo acute hazard—none of the tests shows effect
CLASS IISlight acute hazard—a statistically significant percentage effect is reached in at least one test, but the effect level is below 50%
CLASS IIIAcute hazard—the 50% effect level is reached or exceeded in at least one test, but the effect level is below 100%.
CLASS IVHigh acute hazard—the 100% effect is reached in at least one test
CLASS VVery high acute hazard—the 100% percentage effect is reached in all the tests
Note: Percentage effect: 10% effect = slight toxicity for daphnia and fish; 20% effect = slight toxicity for algae and bacteria; 50% and > effect = toxicity for all test organisms (bacteria, algae, daphnia, and fish).
Table 3. Yeast Estrogen Screen (YES) assay results (average) for samples containing PFOA.
Table 3. Yeast Estrogen Screen (YES) assay results (average) for samples containing PFOA.
Initial Concentration (ng/L)EEQ (ng/L) in the InfluentFinal Concentration (ng/L)EEQ (ng/L) in the EffluentToxicity
1501.94 ± 0.256580.97 ± 0.034No
3000.72 ± 0.065720.99 ± 0.108No
4501.21 ± 0.0701420.23 ± 0.029No
6000.84 ± 0.0372410.51 ± 0.063No
7500.99 ± 0.0964760.89 ± 0.041No
9001.06 ± 0.0825960.66 ± 0.027No
10503.15 ± 0.0567020.84 ± 0.037No
Table 4. Yeast Estrogen Screen (YES) assay results (average) for samples containing PFOS.
Table 4. Yeast Estrogen Screen (YES) assay results (average) for samples containing PFOS.
Initial Concentration (ng/L)EEQ (ng/L) in the InfluentFinal Concentration (ng/L)EEQ (ng/L) in the EffluentToxicity
1501.45 ± 0.038620.97 ± 0.036No
3000.84 ± 0.0651391.16 ± 0.079No
4500.97 ± 0.00341480.64 ± 0.039No
6000.43 ± 0.0364000.89 ± 0.041No
7501.08 ± 0.0926161.06 ± 0.096No
9001.96 ± 0.0867861.06 ± 0.079Yes
10501.11 ± 0.1348110.98 ± 0.043No
Table 5. Results of the Daphnia magna screening assays expressed as percentage mortality after 24 and 48 h in samples containing PFOA.
Table 5. Results of the Daphnia magna screening assays expressed as percentage mortality after 24 and 48 h in samples containing PFOA.
Final Concentration (ng/L)HoursMortality (%)Toxicity Hazard Potential
Control240No acute hazard
480
582450Acute hazard
4850
722450Acute hazard
4850
14224100Very high acute hazard
48100
24124100Very high acute hazard
48100
47624100Very high acute hazard
48100
59624100Very high acute hazard
48100
70224100Very high acute hazard
48100
Table 6. Results of the Daphnia magna screening assays expressed as percentage mortality after 24 and 48 h in samples containing PFOS.
Table 6. Results of the Daphnia magna screening assays expressed as percentage mortality after 24 and 48 h in samples containing PFOS.
Final Concentration (ng/L)HoursMortality (%)Toxicity Hazard Potential
Control240No acute hazard
480
6224100Very high acute hazard
48100
13924100Very high acute hazard
48100
14824100Very high acute hazard
48100
40024100Very high acute hazard
48100
61624100Very high acute hazard
48100
78624100Very high acute hazard
48100
81124100Very high acute hazard
48100
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Kibambe, M.G.; Momba, M.N.B. Ecotoxicological Assessment of Perfluorooctane Sulfonate and Perfluorooctanoic Acid Following Biodegradation: Insights from Daphnia magna Toxicity and Yeast Estrogen Screen Assays. Water 2025, 17, 3368. https://doi.org/10.3390/w17233368

AMA Style

Kibambe MG, Momba MNB. Ecotoxicological Assessment of Perfluorooctane Sulfonate and Perfluorooctanoic Acid Following Biodegradation: Insights from Daphnia magna Toxicity and Yeast Estrogen Screen Assays. Water. 2025; 17(23):3368. https://doi.org/10.3390/w17233368

Chicago/Turabian Style

Kibambe, Muyasu Grace, and Maggy Ndombo Benteke Momba. 2025. "Ecotoxicological Assessment of Perfluorooctane Sulfonate and Perfluorooctanoic Acid Following Biodegradation: Insights from Daphnia magna Toxicity and Yeast Estrogen Screen Assays" Water 17, no. 23: 3368. https://doi.org/10.3390/w17233368

APA Style

Kibambe, M. G., & Momba, M. N. B. (2025). Ecotoxicological Assessment of Perfluorooctane Sulfonate and Perfluorooctanoic Acid Following Biodegradation: Insights from Daphnia magna Toxicity and Yeast Estrogen Screen Assays. Water, 17(23), 3368. https://doi.org/10.3390/w17233368

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