Occurrence of parabens in aquatic environments and sediments and efficiency of wastewater treatment plants in parabens removal

Abstract

Parabens are endocrine-disrupting chemicals, and their entry into the human body via water sources has adverse effects on human health. In the present study, the occurrence of methylparaben, ethylparaben, propylparaben, and butylparaben in three domestic, hospital, and industrial wastewater treatment plants, and in river water and sediments, was investigated. The samples were concentrated via solid-phase extraction and analyzed by high-performance liquid chromatography. Results showed that the recovery percentage for all examined parabens was within the acceptable range of 97.4% to 103.6%, and the highest paraben concentrations were observed in influents from the hospital wastewater treatment plant (methylparaben: 34.4 ± 3.6 µg/L; propylparaben: 30.9 ± 5.4 µg/L). The study indicated that processes used in wastewater treatment plants are unable to completely remove parabens; however, the anaerobic-anoxic-aerobic process has the highest efficiency for paraben removal. The anaerobic-anoxic-aerobic process’s efficiency for removing methylparaben, ethylparaben, propylparaben, and butylparaben was 72.24%, 78.37%, 79.57%, and 72.99%, respectively. Based on the results, the anaerobic-anoxic-aerobic process can be used as a primary treatment to reduce the concentration of emerging contaminants in wastewater. Given the health effects of parabens, future studies should investigate processes capable of completely removing emerging pollutants such as parabens from aquatic environments.

Introduction

In recent decades, the emerging contaminants that have been discharged into water resources have posed a potential threat to human health and have had environmental impacts¹. Urbanization, intensification of manufacturing processes, and population growth have played a significant role in the increase in the level of these pollutants in the water resources². Emerging contaminants have complex chemical structures that are resistant to conventional biological degradation³. So, the discharge of any concentration of emerging contaminants can reduce the water quality and disrupt conventional and natural treatment^4,5. Although emerging pollutants include a wide range of health-threatening contaminants, some of them, including parabens, bisphenol A, phthalates, per- and polyfluoroalkyl substances (PFAS), pharmaceuticals and personal care products (PPCPs), atrazine, and glyphosate, can disrupt the endocrine system^6,7.

Parabens are endocrine-disrupting chemicals (EDCs) that, owing to their antifungal and antibacterial properties, can extend the shelf life of products⁸. Parabens can disrupt hormonal balance by mimicking hormone structures⁹. Parabens can disrupt endocrine functioning even at low concentrations, thereby increasing the risk of breast cancer¹⁰. Parabens can be absorbed through the gastrointestinal tract and via drinking water and can cause immunotoxicity, oxidative stress, and genotoxicity¹¹. Parabens can enter and accumulate in aquatic environments due to their environmental stability, high water solubility, and resistance to complete removal in conventional wastewater treatment¹². Additionally, parabens have low vapor pressures (9.29 × 10^{− 5}-1.86 × 10^{− 4} mm Hg), indicating moderate volatility, and volatilization from water surfaces is not expected^13,14. The health and environmental impacts of parabens indicate that their presence in aquatic environments is a critical concern.

The discharge of wastewater that contains parabens into water resources can directly impact the water quality by increasing the concentration of parabens and other emerging contaminants¹⁵. Studies have indicated that parabens are present in the urine, so wastewater is one of the potential sources of these emerging contaminants, which can induce adverse effects on the water resources¹⁶. According to hydrophobicity properties, paraben molecules can be adsorbed onto sediments or suspended solids, which are in the wastewater and water streams^17,18. Among the parabens, methylparaben (MePB) and propylparaben (PrPB) are most used as preservatives and can be discharged from industrial wastewater into water resources^1,13,19.

The toxicological findings underscore the need for continued monitoring of the presence and concentrations of parabens in aquatic environments to inform management decisions¹². In many countries, including Iran, water stress has recently prompted governments to consider the reuse of wastewater as a potential source of water supply²⁰. Because conventional wastewater treatment methods are unable to remove EDCs such as parabens, monitoring wastewater for their presence is essential for informed management decisions²¹. The present study aimed to investigate (1) the measurement of the concentrations of MePB, ethylparaben (EtPB), PrPB, and butylparaben (BuPB) in the influent and effluent of domestic, hospital, and industrial wastewater treatment plants (WWTPs); and (2) the concentration of parabens in downstream, upstream, and the sediments of a river as a water supplier.

Materials and methods

Reagents and standards

In the present study, the analytical standard of used reagents includes MePB (C₈H₈O₃, 99.7%), EtPB (C₉H₁₀O₃, 99%), BuPB (C₁₁H₁₄O₃, ≥ 99.0%), and PrPB (C₁₀H₁₂O₃, ≥ 99.0%), which were purchased from Sigma-Aldrich (St. Louis, MO, USA). In all sample preparations, ultrapure water (Milli-Q, 18.2 MΩ cm^{− 1} conductivity) was used. Because the solubility of parabens in water is low (the solubility of MePB, EtPB, PrPB, and BuPB were 0.25%, 0.17%, 0.05%, and 0.02%, respectively), the standard stock solutions of MePB, EtPB, PrPB, and BuPB (1 mg/mL) were prepared separately in methanol of high-performance liquid chromatography (HPLC) grade, which was purchased from Sigma-Aldrich (St. Louis, MO, USA) and stored at 4 °C. All other chemical substances used in the procedure were of analytical grade and obtained from Sigma-Aldrich (St. Louis, MO, USA). The physicochemical properties of wastewater and river water samples, including pH, temperature, chemical oxygen demand (COD), biochemical oxygen demand (BOD), total suspended solids (TSS), and total dissolved solids (TDS), were measured in accordance with the standard procedures described by Abu Bakar et al. (2020)²².

Instrumentation and chromatographic conditions

HPLC (YL9100, Younglin, Korea) equipped with an autosampler (YL 9150), vacuum degasser unit, quaternary pump, column, and UV/Vis detector was used to detect the concentration of MePB, EtPB, BuPB, and PrPB in the samples. The ZORBAX SB-C18 (150 mm × 4.6 mm, 5 μm) was used as the stationary phase at 22 °C (room temperature), and the mobile phase solutions were (A) MeOH and (B) 1% acetic acid in ultrapure water, which MeOH is the main solvent and 1% acetic acid that prepared in ultrapure water, is the mobile phase modifier^23,24. The flow rate of the two mobile phases, consisting of MeOH and acetic acid (1%) (50:50), was 1 mL/min for elution in isocratic mode²⁵. Additionally, the injected volume was set to 10 µL, and the concentrations of MePB, EtPB, BuPB, and PrPB were measured at 254 nm²⁶.

Method validation

Quality assurance and quality control (QA/QC)

In the present study, 1, 3, and 5 µg/L of MePB, EtPB, BuPB, and PrPB were spiked into a blank to conduct rigorous quality assurance and quality control (QA/QC) procedures; this process was carried out to ensure the accuracy, precision, and reliability of the analytical data. In the present study, Eqs. 1 and 2 were used to calculate the Limit of Detection (LOD) and Limit of Quantification (LOQ), where σ denotes the standard deviation (SD) of the response, and S denotes the slope of the calibration curve for each paraben compound. These parameters can be used to assess the analytical method’s sensitivity and reliability. LOD and LOQ define the lowest concentrations of an analyte that can be reliably detected and quantified, respectively²⁷.

1

2

In the present study, all samples were collected and processed in strictly glassware to prevent cross-contamination. Glassware was washed with paraben-free detergents, rinsed with high-purity solvents, and dried at 400 ˚C before sampling. Laboratory reagent blanks and field blanks were analyzed with every batch of 10 samples to monitor for background contamination. Also, the analysis indicated that no parabens were detected in any blank samples above the LOD.

Calibration curve

The prepared stock solutions of MePB, EtPB, BuPB, and PrPB were used to prepare standard working solutions, and then the calibration curves were prepared using 7 concentration levels ranging from 0.5 to 20 µg/L in triplicate for all parabens. Matrix-matched calibration was employed to compensate for potential matrix effects (ME) common in complex wastewater and sediment samples. Standard solution concentrations were spiked into samples; then the linear curve for each paraben was drawn, plotting the area of the observed peak against concentration. The concentrations of MePB, EtPB, BuPB, and PrPB were measured at 254 nm, and calibration curves were constructed based on the response signals²⁸.

Sample collection

In the present study, 27 samples were collected from the influent and effluent of 3 full-scale WWTPs (domestic, hospital, and industrial) and a river (downstream, upstream, and sediments) located in Tehran, Iran, in July 2025. The processes used in domestic, hospital, and industrial WWTPs were the anaerobic-anoxic-aerobic (A₂O), moving-bed biofilm sequencing batch reactor (MBSBR), and conventional activated sludge (CAS), respectively. The samples collected from each sampling point (9 points) in amber glass bottles (5 L) were then transferred to the lab in a cooling box and stored at 4˚C. In the next step, the collected samples were filtered through 0.22 μm cellulose acetate (CA) membrane filters (Whatman, Maidstone, UK) and stored at 4 °C in the dark for analysis. The collected samples were analyzed in triplicate^29,30.

Preparation and analysis

The MePB, EtPB, PrPB, and BuPB were extracted from aquatic environments and sediments by solid-phase extraction (SPE). The paraben compounds in the aqueous water samples (wastewater and river) were extracted by SPE cartridges (Oasis HLB, 6 mL and 200 mg)³¹. Because the adsorbents should be activated in the SPE method, SPE cartridges are preconditioned with 5 mL of MeOH, 5 mL of ultrapure water, and 5 mL of acidified ultrapure water (pH = 2, 2 N HCl). Because the efficiency of paraben extraction under acidic conditions is higher, samples were acidified to pH 3 with HCl before SPE loading¹². Additionally, lyophilization was performed on sediment samples. So, 20 g of sediment samples is transferred to a baker, exposed to a mixed solution of MeOH: acetonitrile (50:50, v/v), and sonicated for 10 min. After that, the solution should be centrifuged (6000 rpm, 5 min)^32,33. Finally, the supernatant was diluted with acidified water to adjust the pH to 3 before SPE loading. After loading 500 mL of filtered (0.45 μm) sample at 1 mL/min, the cartridge was washed with 10 mL of ultrapure water and dried under vacuum for 5 min³⁴. The parabens were eluted with 10 mL of MeOH and 10 mL of MeOH: acetonitrile (50:50, v/v) and concentrated by a gentle stream of nitrogen (40˚C). Reconstituted from the extracted compounds carried out in 5 mL of ultrapure water: acetonitrile (40:60, v/v) and concentrated to 1 mL solution. Finally, prepared samples filtered through a 0.22 μm CA membrane (Whatman, Maidstone, UK) and stored at -20 °C were analyzed by HPLC-UV-Vis¹².

Statistical analysis

In the present study, R software (v 3.6.0, Missouri, USA) and Microsoft Excel 2016 were used to carry out the statistical analysis. All experimental data are presented as mean ± SD of triplicate measurements. The normality of the data distribution was assessed by the Shapiro-Wilk test. A one-way analysis of variance (ANOVA) followed by Tukey’s HSD post hoc test was used to determine significant differences in paraben concentrations among the WWTPs, and t-tests were used to compare two specific groups. A p-value < 0.05 was considered statistically significant for all tests.

Results and discussion

Physicochemical properties of wastewater and river water samples

The values of pH, temperature, COD, BOD₅, TSS, and TDS of influent and effluent samples from domestic, industrial, and hospital WWTPs, as well as upstream and downstream river water, are presented in Table 1.

Table 1.

The physicochemical properties of domestic, hospital, and industrial wastewater and river water samples.

Sampling location		pH	Temperature (°C)	COD (mg/L)	BOD5 (mg/L)	TSS (mg/L)	TDS (mg/L)
Domestic WWTP	Influent	7.5 ± 0.2	24.8 ± 1.7	428 ± 45	195 ± 28	268 ± 32	712 ± 85
	Effluent	7.6 ± 0.1	23.9 ± 1.4	58 ± 14	24 ± 5	22 ± 6	305 ± 42
Industrial WWTP	Influent	6.1 ± 0.4	26.5 ± 2.3	712 ± 68	248 ± 35	385 ± 52	1350 ± 165
	Effluent	6.9 ± 0.3	25.4 ± 1.8	245 ± 32	72 ± 16	48 ± 11	820 ± 110
Hospital WWTP	Influent	7.2 ± 0.3	25.6 ± 1.9	652 ± 78	378 ± 42	312 ± 38	825 ± 95
	Effluent	7.5 ± 0.2	24.7 ± 1.6	58 ± 14	19 ± 4	26 ± 7	418 ± 68
River	Upstream	7.8 ± 0.1	14.2 ± 1.3	12 ± 2	5 ± 1	10 ± 3	138 ± 25
	Downstream	7.7 ± 0.2	17.8 ± 1.5	26 ± 3	13 ± 3	31 ± 7	225 ± 22

Calibration curves and validity of method

As shown in Fig. 1a, calibration curves are highly reliable (R² > 0.99) for quantitative analysis in real samples. A standard mixed solution of MePB, EtPB, BuPB, and PrPB, each at a concentration of 10 µg/L, was prepared and injected into the HPLC. The chromatogram of the standard mixture of parabens is shown in Fig. 1b and indicates retention times of 7.14 min, 9.79 min, 11.45 min, and 15.61 min for MePB, EtPB, BuPB, and PrPB, respectively. The regression equations were obtained from the slopes and intercepts of the calibration curves (Fig. 1a) for parabens. They can be used to determine the concentrations of MePB, EtPB, BuPB, and PrPB in real samples from the areas of the corresponding peaks in the chromatogram.

Fig. 1.

Calibration curves of MePB, EtPB, PrPB, and BuPB (a), and chromatogram of parabens in mixed solution (b).

Table 2 presents the R² values of the calibration curves, LOD, and LOQ for MePB, EtPB, PrPB, and BuPB concentrations that can be detected via HPLC-UV-Vis in aquatic and sediment samples.

Table 2.

LOD and LOQ for the analysis of parabens using HPLC-UV-Vis.

Analyte	R 2	LOD (µg/L)	LOQ (µg/L)
MePB	0.9969	0.15	0.45
EtPB	0.9913	0.44	1.35
PrPB	0.9948	0.19	0.60
BuPB	0.9922	0.34	1.03

Table 3 shows that the selected methodology for detecting paraben concentration has high accuracy and negligible systematic bias, as the recovery percentage (R%) for all examined parabens was within the acceptable range of 97.4% to 103.6%. The relative standard deviation (RSD%) can be used to evaluate the precision of the methodology and is an index of the reproducibility of the detection method. In the present study, the RSD ranged from 1.36% to 5.58%, indicating satisfactory reproducibility of the technique.

Table 3.

Accuracy and precision of the proposed method.

Analytes	Spiked concentration (µg/L)	R (%)	RSD (%)
MePB	1	102.3 ± 1.7	1.66
	3	99.1 ± 2.2	2.22
	5	102.1 ± 5.7	5.58
EtPB	1	101.9 ± 3.6	3.53
	3	103.6 ± 4.1	3.96
	5	99.6 ± 3.5	3.51
PrPB	1	98.4 ± 2.3	2.34
	3	103.3 ± 1.4	1.36
	5	97.4 ± 2.9	2.98
BuPB	1	99.2 ± 1.6	1.61
	3	100.8 ± 3.6	3.57
	5	99.7 ± 2.7	2.71

The level of parabens in the wastewater samples

Table 3 presents the mean concentration of parabens in the influent and effluent of domestic, hospital and industrial WWTPs. Results showed that the highest concentration of MePB, EtPB, PrPB, and BuPB was detected in the influent of hospital WWTPs, which reflects the use of paraben-containing pharmaceuticals and antiseptics in the patient treatment process³⁵. The high concentrations of parabens such as MePB in hospital wastewater are consistent with the widespread use of these compounds as preservatives in pharmaceuticals and PPCPs. The dominance of MePB and PrPB over other EDCs aligns with global trends reported in literature, such as studies in China³⁶ and the United States³⁷. The significantly higher levels in hospital wastewater compared to domestic wastewater highlight hospitals as major point-source contributors of EDCs to municipal wastewater systems³⁸. The concentration of parabens and other emerging contaminants in the industrial wastewater is strongly dependent on the type of industry because wastewater is a chemical fingerprint of the industrial activity and the wastewater of various industries cannot be same³⁹.

In the present study, samples were collected from industrial WWTPs designed for the pharmaceutical, food, and cosmetic industries. As presented in Table 4; Fig. 2, the mean (SD) concentration of MePB, EtPB, PrPB, and BuPB in the influent of industrial WWTPs was 26.9 (± 1.8) µg/L, 21.4 (± 2.8) µg/L, 15.3 (± 1.5) µg/L, and 9.2 (± 2) µg/L, respectively. Ramírez et al. (2012) investigated the concentrations of parabens in three industrial WWTPs; the results showed that, unlike in the present study, PrPB had a higher concentration than MePB, which could be due to differences in industry type⁴⁰. Table 4; Fig. 2 show that the concentration of parabens in domestic wastewater was much lower than that of hospital and industrial wastewater. One reason for this difference may be that the volume of wastewater entering domestic treatment plants is much greater than that entering industrial and hospital treatment plants, thereby diluting and reducing concentrations.

Table 4.

Mean concentration of parabens in aquatic environments and sediments.

Sampling location		MePB (µg/L)		EtPB (µg/L)		PrPB (µg/L)		BuPB (µg/L)
		Mean	SD	Mean	SD	Mean	SD	Mean	SD
Domestic Wastewater	Influent	23	5.65	14.8	3.4	9.2	2.1	10.4	2.9
	Effluent	6.3	1.1	3.2	0.5	1.9	0.5	2.8	0.7
Industrial Wastewater	Influent	26.9	1.8	21.4	2.8	15.3	1.5	9.2	2
	Effluent	9.9	2.6	7.6	0.9	5.35	1.1	3.4	0.45
Hospital Wastewater	Influent	34.4	3.6	30.9	5.4	18.95	1.8	12.45	1.1
	Effluent	13	3	11.5	2	7	0.8	4	0.1
River	Upstream	0.3	0.1	ND*	-	ND	-	ND	-
	Downstream	1	0.1	ND	-	0.45	0.3	ND	-
	Sediments**	2	0.2	0.7	0.4	ND	-	3.5	0.5

*ND: Not detected (lower than LOD) ** µg/kg.

Fig. 2.

The clustered histogram of average concentration of parabens across all (a) domestic, hospital and industrial wastewaters, and (b) the river water and sediment samples.

The level of parabens in the river

The level of parabens in the water samples

Table 4 shows that in the collected samples from upstream of the river, the concentration of MePB was 0.3 µg/L, and the concentrations of other parabens were below the LOD and could not be detected by HPLC. So, MePB is the predominant paraben entering the upstream of the river, likely originating from diffuse sources like agricultural runoff or natural attenuation processes⁴¹. Additionally, the detection of MePB in the upstream can reveal patterns in stormwater and rural effluents, in which shorter-chain variants predominate due to their higher solubility and lower sorption to sediments⁴². Penrose and Cobb (2023) measured paraben concentrations in the Brazos River in Texas. Their results showed that the concentration of MePB in the upstream of the Brazos River was 0.00083 µg/L⁴³, which was lower than the concentration of MePB in the upstream (0.3 µg/L) in the present study.

As presented in Table 4, the concentration of MePB and PrPB increased in the collected samples from downstream of the river. Previous studies have indicated that the concentration of parabens in the upstream region is 50% lower than in the downstream area. This increase may be attributable to wastewater discharges and human activities in the downstream region⁴⁴. Table 3 presents that the mean (SD) concentration of MePB in collected water samples from upstream of the river was 0.3 (± 0.07) µg/L, which increased to 1 (± 0.1) µg/L downstream of the river, while the mean (SD) concentration of PrPB changed from non-detectable (ND) levels upstream to 0.45 (± 0.3) µg/L downstream. However, EtPB and BuPB concentrations were lower than LOD in the upstream and downstream of the river. Parabens are emerging contaminants that can enter aquatic environments via point and non-point sources⁴⁵. In the present study, increases in MePB and PrPB concentrations downstream of the river are attributed to anthropogenic activities, such as industrial wastewater discharges. These elevated levels result from the incomplete removal of parabens in treatment processes, combined with constant domestic inputs from cosmetics and personal care products, low river dilution capacity, and the moderate persistence of these compounds in the aquatic environment. The study by Penrose and Cobb (2023) was consistent with the present study and confirmed that paraben concentrations were higher downstream than upstream, likely due to the introduction of various pollutants along the Brazos River⁴³. Feng et al. (2019) found that MePB and PrPB were the most abundant in surface water, which is consistent with the results of the present study⁴⁶.

The level of parabens in the sediments

As shown in Table 4, the concentrations of parabens in the river’s sediments differed markedly from those in water samples. Results showed that the highest detected concentration was for BuPB (3.5 ± 0.5 µg/kg dw); the mean (± SD) concentrations of MePB and EtPB were 2 (± 0.2) and 0.7 (± 0.4) µg/kg dw, respectively; and PrPB levels were below the LOD. Table 3; Fig. 2 show that the highest paraben concentrations in the sediments are associated with BuPB. The octanol-water partition coefficient (log K_ow) is a key physicochemical parameter that quantifies the hydrophobicity of parabens and directly affects their environmental behavior⁴⁷. An increase in log Kow enhances the partitioning of parabens from water into sediments, thereby increasing their concentration in sediments⁴⁸. BuPB, as a longer-alkyl-chain parabens, has a higher log K_ow (3.57) than PrPB (3.04), EtPB (2.47), and MePB (1.96)⁴⁹. Therefore, BuPB is more likely to accumulate in sediments than other parabens. The results show that the levels of BuPB in collected water samples from upstream and downstream of the river were lower than the LOD, while the mean (SD) concentration of BuPB in sediment samples was 3.5 (± 0.5) µg/kg dry weight, indicating that BuPB can accumulate in river bottom sediments over the long term due to its high log K_ow^13,49. The detection of parabens in river sediments indicates that they exhibit long-term accumulation⁵⁰. As mentioned, parabens are relatively hydrophobic, they tend to accumulate into organic-rich sediments⁴⁷. This is environmentally significant because even if aqueous concentrations in the river are low, the sediments act as a secondary source of exposure for benthic organisms. The toxicological studies indicate that, presence of these EDCs in the aquatic environment, even at ng/L levels, poses a potential risk to the endocrine systems of aquatic fauna^50,51.

The effect of the WWTP process on the removal of parabens

After measuring the concentrations of MePB, EtPB, PrPB, and BuPB in various aquatic environments, the removal efficiency of WWTPs should be evaluated to identify the most effective treatment method to minimize effluent contamination. Table 5 presents the efficiency of the A₂O, CAS, and MBSBR processes in removing MePB, EtPB, PrPB, and BuPB from wastewater. Results showed that the efficiency of the A₂O process for removal of MePB, EtPB, PrPB, and BuPB were 72.24%, 78.37%, 79.57%, and 72.99%, respectively. The efficiency of the CAS process for removal of MePB, EtPB, PrPB, and BuPB were 63.48%, 64.4%, 65.18%, and 63.04%, respectively. Additionally, the MBSBR can remove 62.15%, 62.7%, 61.7%, and 67.5% of MePB, EtPB, PrPB, and BuPB, respectively. The efficiency of WWTPs is directly related to the chemical properties of the pollutants in the wastewater. Because pollutant behavior varies across wastewater treatment systems, the efficiency of each treatment method should be evaluated separately to identify the most cost-effective process⁵².

Table 5.

The efficiency of A₂O, CAS, and MBSBR processes in the removal of MePB, EtPB, PrPB, and BuPB from wastewater.

Sampling location	MePB Removal %	EtPB Removal %	PrPB Removal %	BuPB Removal %
A2O	70.11	76.90	81.14	68.9
	72.38	78.05	78.19	76.63
	74.23	80.17	79.38	73.46
CAS	69.53	62.77	61.29	59.75
	65.36	66.59	69.89	63.47
	55.56	63.95	64.34	65.89
MBSBR	67.14	64.06	57.02	66.96
	64.36	60.74	65.09	65.97
	54.96	63.49	63.07	69.81

The efficiency of WWTPs’ process on the removal of MePB

Figure 3 presents box plots of the efficiency of the A₂O, CAS, and MBSBR processes, along with a statistical comparison of MePB removal. According to Fig. 3a, the median removal of MePB by A₂O, CAS, and MBSBR was 72.4%, 65.4%, and 64.4%, respectively. Results showed that the narrowest interquartile ranges (IQRs) were for A2O (IQRs = 71.3–73.3%), which can indicate consistency⁵³. Also, Fig. 3a shows that the IQR ranges for CAS (IQRs = 60.5–67.5%) and MBSBR (IQRs = 59.7–65.8%) are broader. The homogeneity of variance in MePB removal by A₂O, CAS, and MBSBR was confirmed by the Fligner-Killeen test (p-value = 0.46), which justifies ANOVA (see Table 4). As presented in Fig. 3b, the one-way ANOVA shows that the change in the treatment method for the removal of MePB is not significant (p-value = 0.138), although the nominal mean of MePB removal was higher in the A₂O (72.2%). As mentioned, A₂O compared to CAS and MBSBR has a higher nominal mean of MePB removal, which may be related to the low hydrophobicity (log Kow ≈ 1.9) of this compound, which can enable hydrolysis by esterase enzymes across the aerobic process, where, in this condition, the ester bond is broken, and p-hydroxybenzoic acid and methanol are produced^54,55.

Fig. 3.

The box plot of efficiency (a) and statistical comparison of (b) of MePB removal efficiency across A₂O, CAS, and MBSBR processes.

The efficiency of WWTPs’ process on the removal of EtPB

The box plot of EtPB removal efficiency via the A₂O, CAS, and MBSBR processes, along with a statistical comparison, is shown in Fig. 4. According to Fig. 4a, the median EtPB removal efficiencies were 78.06%, 63.95%, and 65.50% for A₂O, CAS, and MBSBR, respectively. Also, the highest median EtPB removal was observed in the A₂O process. Wang and Kennan (2016) investigated the fate of parabens in WWTPs in New York; their results indicate that the median removal of EtPB in the A2O process was 72%-85%, consistent with the results of the present study³⁷. Previous studies have shown that the CAS process’s efficiency in removing parabens varies widely, from 56% to 99%. Haman et al. (2015) reported that the CAS process’s efficiency for removing EtPB was 60–70%, consistent with the results of the present study¹³. Results showed that the IQRs for A₂O (IQRs = 76.9-80.17%), CAS (IQRs = 62.78–66.59%), and MBSBR (IQRs = 60.74–64.07%) were narrow, so the reproducibility of experiments is high, with little scatter due to random errors, which strengthens the statistical power of the ANOVA results and supports the conclusion that A₂O’s superiority is not confounded by high process noise. As presented in Table 4, the Fligner-Killeen test (p-value = 0.91) confirmed homogeneity of variances across the A₂O, CAS, and MBSBR processes for EtPB removal, thereby supporting the assumptions of the parametric analysis. The one-way ANOVA indicates that process changes have a significant effect on EtPB removal from wastewater (p < 0.001). Figure 4b shows the post-hoc Tukey HSD tests of EtPB removal across A₂O, CAS, and MBSBR. According to this test, the efficiency of the A₂O process was significantly higher than that of CAS and MBSBR (p < 0.001), whereas no significant difference was observed between CAS and MBSBR (p = 0.528). Biological processes can effectively remove EtPB in the initial stage and reduce the pollution load; advanced EtPB removal processes, which are high-efficiency but also high-cost, can be used to treat wastewater with lower EtPB concentrations^56,57.

Fig. 4.

The box plot of efficiency (a) and statistical comparison of (b) of EtPB removal efficiency across A₂O, CAS, and MBSBR processes.

The efficiency of WWTPs’ process on the removal of PrPB

The efficiency of A₂O, CAS, and MBSBR processes in the removal of PrPB and statistical comparison is shown in Fig. 5. According to Fig. 5a, A₂O has the highest efficiency for PrPB removal. As presented in Fig. 5a, the A₂O process exhibited the highest median PrPB removal efficiency (79.38%), with a narrow interquartile range (IQR = 1.48%) indicating low variability among replicates. Wang and Kannan (2016) found that the A₂O process removed 81.2%-97.5% of PrPB in wastewater, which is comparable to the efficiency of A₂O in the present study³⁷. In comparison, CAS and MBSBR displayed lower medians (64.35% and 63.07%, respectively) and wider IQRs (4.30% and 4.04%, respectively). The statistical analysis confirmed that the variances of the removal efficiency have homogeneity (p-value = 0.51), and one-way ANOVA indicated a significant effect (p-value = 0.002) of process type on PrPB removal efficiency (Table 4). The results of Lu et al. (2018) present that the CAS process has a moderate removal for PrPB, and they can be used to reduce the concentration before the advanced treatment⁵⁸. Figure 5b presents the post hoc Tukey HSD tests for the removal of PrPB using the A₂O, CAS, and MBSBR processes. Results showed that the A₂O process was significantly more efficient than CAS and MBSBR (p = 0.007). In contrast, no significant difference was observed between CAS and MBSBR in the removal of PrPB from wastewater (p = 0.509).

Fig. 5.

The box plot of efficiency (a) and statistical comparison of (b) of PrPB removal efficiency across A₂O, CAS, and MBSBR processes.

The efficiency of WWTPs’ process on the removal of BuPB

The efficiency of wastewater treatment processes in removing BuPB, along with a statistical comparison, is shown in Fig. 6. As with other parabens, the highest efficiency for BuPB removal was observed in the A2O process (Fig. 6a). As shown in Fig. 6a, the median BuPB removal values for A₂O, CAS, and MBSBR were 73%, 67%, and 63%, respectively. The A₂O process, due to its integrated anaerobic-anoxic-aerobic design, demonstrates the highest efficiency in removing BuPB from wastewater. This design fosters greater microbial diversity and extends retention times, both of which are beneficial for the hydrolytic and oxidative degradation of resistant alkyl esters such as BuPB⁵⁹. Amin et al. (2019) investigated the efficiency of the CAS process for removing parabens from wastewater in Isfahan, Iran. Their results indicate that CAS can remove 62% of BuPB from sewage, which is slightly lower than the findings of the present study; these differences may be attributed to differences in wastewater characterization and operational techniques across the two WWTPs⁵⁹. The homogeneity of variances for the removal of BuPB via A₂O, CAS, and MBSBR was tested by the Fligner-Killeen test. Results confirmed that the variances in removal efficiency were homogeneous (p-value = 0.55). A one-way ANOVA of removal efficiency showed a statistically significant effect of process type (p-value = 0.021), suggesting that treatment type substantially influences BuPB removal (see Table 6). Since the ANOVA is substantial, pairwise comparisons must be made to determine which specific pairs of processes have created this overall variance, which is determined using the post-hoc Tukey HSD tests and is shown in Fig. 6b. Results showed that the efficiency of the A₂O process was significantly (p-values = 0.007) greater than CAS and MBSBR. In contrast, no significant difference was observed between CAS and MBSBR in the removal of BuPB from wastewater (p = 0.509). As shown in Fig. 6b, the A2O process has significantly higher BuPB removal efficiency than CAS (p-value = 0.018). This demonstrates A₂O’s superior performance in removing BuPB. There were no significant differences observed between A₂O and MBSBR (p-value = 0.16). Similarly to other parabens, no significant difference (p = 0.248) was observed between CAS and MBSBR in the removal of BuPB from wastewater.

Fig. 6.

The box plot of efficiency (a) and statistical comparison of (b) of BuPB removal efficiency across A₂O, CAS, and MBSBR processes.

Table 6.

The statistical analysis of the efficiency of the A₂O, CAS, and MBSBR processes in the removal of parabens.

Paraben Type	Fligner-Killeen test
	Chi-squared		Degree of freedom (DF)			p-value
MePB	1.517		2			0.46
EtPB	0.172		2			0.91
PrPB	1.325		2			0.51
BuPB	1.174		2			0.55

ANOVA
Paraben Type	Parameters	DF	Sum Square	Mean Square	F-value	p-value
MePB	WWTPs type	2	180.3	90.13	2.802	0.138
	Residual	6	190	32.16
EtPB	WWTPs type	2	440.7	220.34	68.06	< 0.001
	Residual	6	19.4	3.24
PrPB	WWTPs type	2	537.2	268.61	20.74	0.002
	Residual	6	77.7	12.95
BuPB	WWTPs type	2	149.17	74.58	7.8	0.021
	Residual	6	57.34	9.56

Tukey HSD
Paraben Type	WWTPs combination	different	Lower	Upper		p-value
MePB	A2O-CAS	-8.75	-22.96	5.44		0.221
	A2O-MBSBR	-10.08	-24.29	4.11		0.153
	CAS-MBSBR	-1.32	-15.53	12.87		0.955
EtPB	A2O-CAS	-13.93	-18.44	-9.42		< 0.001
	A2O-MBSBR	-15.6	-20.11	-11.1		< 0.001
	CAS-MBSBR	-1.67	-6.17	2.83		0.52
PrPB	A2O-CAS	-14.39	-23.4	-5.37		0.006
	A2O-MBSBR	-17.83	-26.85	-8.82		0.002
	CAS-MBSBR	-3.44	-12.46	5.56		0.509
BuPB	A2O-CAS	-9.95	-17.7	-2.21		0.017
	A2O-MBSBR	-5.41	-13.15	2.32		0.16
	CAS-MBSBR	4.54	-3.19	12.28		0.24

The removal of parabens by the A₂O process is governed by microbial biodegradation and physical adsorption onto activated sludge. The MePB and EtPB, as shorter-chain parabens, are highly susceptible to enzymatic hydrolysis⁶⁰. The removal efficiencies of parabens in WWTPs show that while conventional biological treatments, such as the A₂O process, are effective, they cannot completely remove them. The primary removal mechanism for shorter-chain parabens (MePB and EtPB) is likely rapid biodegradation mediated by the prbA gene-coding esterase found in various bacteria, but the removal of longer-chain parabens like BuPB is more complex⁶¹. Due to its log K_ow, BuPB exhibits a greater affinity for the solid phase. Therefore, its removal in the A₂O system is a combination of biodegradation in the aerobic zone and adsorption onto the return activated sludge.

Limitation

The analytical method used in this study has a detection limit of 0.1 µg/L, which is a significant limitation, especially in detecting lower concentrations of parabens in aquatic environments such as surface waters. This limitation could result in concentrations below 0.1 µg/L not being detected by the current method. Therefore, the absence of parabens in these samples cannot be definitively interpreted as their complete absence, but rather as a potential consequence of the sensitivity limitations of the method. To address this issue, future studies could consider more sensitive analytical techniques such as ultra-high-performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS), which could improve detection at lower concentrations and provide a more comprehensive understanding of the distribution of parabens in aqueous environments.

Conclusion

This study has provided essential insights into paraben concentrations in aquatic environments and sediments, and into the efficiency of the A₂O, CAS, and MBSBR processes for removing MePB, EtPB, PrPB, and BuPB. The highest concentration of parabens was detected in the influent of hospital WWTPs, which can be related to the discharge of pharmaceutical residuals into wastewater. Parabens exhibit bioaccumulation; consequently, their concentrations were higher downstream of the river than upstream. Increasing the concentration of parabens in the downstream of a vital water supply can increase adverse effects on human health. The highest log Kow is for BuPB; given its high hydrophobicity, BuPB is more likely to accumulate in sediments than other parabens. In river sediments, BuPB exhibited the highest accumulation (3.5 ± 0.5 µg/kg dry weight), followed by MePB (2.0 ± 0.2 µg/kg dw) and EtPB (0.7 ± 0.4 µg/kg dw), while PrPB was not detected. The A₂O process demonstrated the highest removal efficiencies: 72.24% for MePB, 78.37% for EtPB, 79.57% for PrPB, and 72.99% for BuPB. In contrast, the CAS process achieved 63.48–65.18%, and the MBSBR achieved 61.7–67.5%. According to ANOVA for EtPB, PrPB, and BuPB, changes in the treatment process significantly affect their removal from wastewater (p < 0.05). Results indicate no significant difference between CAS and MBSBR in the removal of parabens from sewage, and that, when the target contaminant is a compound such as a paraben, economic criteria play a primary role in process selection. The study indicated that the processes used in WWTPs are unable to completely remove parabens. Such a failure can harm the receiving water ecosystem and increase downstream paraben concentrations, which can adversely affect human health if the downstream water is used for drinking. Therefore, it is recommended that water suppliers always locate water collection points upstream of rivers. Future studies should be developed the novel methods which can remove emerging contaminants from aquatic solutions in short time from the full-scale WWTPs.

Author contributions

Mohammad Rezvani Ghalhari: Conceptualization, Investigation, Methodology, Formal analysis, Writing - original draft; Babak Shokri: Supervision, Project administration, Funding acquisition, Writing - review & editing; Mohammad Hadi Dehghani: Validation, Data curation, Visualization; Ramin Nabizadeh: Resources, Software, Formal analysis; Mohammadreza Khani: Validation, Data curation, Visualization; Kamyar Yaghmaeian: Supervision, Project administration, Funding acquisition, Writing - review & editing.

Funding

This work is based on research funded by the Iran National Science Foundation (INSF) under project No.4033816. Additionally, the Deputy for Research at Tehran University of Medical Sciences supported this work [grant number 70190].

Data availability

All data generated or analyzed during this study are included in this published article.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

IR.TUMS.SPH.REC.1402.300.

Consent to Participate

Not applicable.

Consent to Publish

Not applicable.