Estrogens are one of the micro-pollutants in the wastewater which have detrimental effects on water living organ- isms. The aim of this study was to evaluate the efficiency of ultrasound to reduce the estrogen (E1) and 17 beta- estradiol (E2) from municipal wastewater. Hence, a cylindrical batch reactor was designed. The effects of powers, frequency, exposure time and pH on reduction efficiency were investigated. The residual concentration of E1 and E2 hormones was measured in reactor effluent by electrochemiluminescence (ECL) method. The results showed that ultrasound removed 85–96% of both E1 and E2 hormones after 45 min while other parameters changes in the range of their operations. Also, the frequency and power of ultrasound had a significant effect on reduction efficiency of hormones while the exposure had no significant effect. Furthermore, the interaction of power and frequency reduced their efficacy to 64.3% (P = 0.005). The result also indicated that the ultrasound waves have high ability to reduce value Steroid hormones from municipal wastewater. The proposed method can be considered as one of the significant strategies for reduction or destruction of hormones from wastewater due to the non-generation of dangerous by- products and the low energy consumption. Keywords: Estrogens, Ultrasound, Wastewater, Reduction, Hormones Introduction 1980 that the detrimental impacts of these hormones Estrogens are one of the micro-pollutants in wastewater were confirmed on fish growth (Behera et al. 2011). Ster- which have detrimental effects on water living organisms oid hormones are endocrine-disrupting compounds in (Azimi et al. 2017; Hamid and Eskicioglu 2012). These the body which have become one of the major concerns hormones are divided into five types: Progestin (Proges - as wastewater effluent in the environment because of terone), Glucocorticosteroids (Cortisol), Mineral corti- negative effects on human health, animals, and ecosystem costeroids (Aldosterone), Androgen (Testosterone), and balance (Aker et al. 2016; Mendoza et al. 2016). Estrogen Estrogen (Nagarnaik et al. 2010). Estrone (E1) and 17 in very low concentrations (less than 0.1 ng/L) interferes beta-estradiol (E2) are the most important estrogen hor- with reproduction of human, livestock, and wildlife and mones in wastewater which are excreted by all humans has a stimulatory effect on breast tumor growth (Ravin - and animals (Guedes-Alonso et al. 2014). Estriol (E3) and dran et al. 2016). Some studies have shown its effect on 17-alpha-ethinyl estradiol (E4) are other estrogens which uterine cancer, ovary and other cancers (Yi et al. 2016). are found in smaller amounts in wastewater (Blair et al. These hormones either are produced naturally in the 2013). The existence of these compounds in wastewater human and animal body or are found in some materials was firstly reported in 1965 (Hamid and Eskicioglu 2012) that humans deal with them on a daily basis. Detergents, but was not seriously investigated by researchers until shampoos, lotions, and cosmetics are new sources of these hormones in environment and wastewater. The levels of estrogens are increasing in municipal *Correspondence: email@example.com; and industrial wastewater due to increasing usage of firstname.lastname@example.org these compounds (Cedat et al. 2016; González et al. Faculty of Chemical and Materials Engineering, Shahrood University 2015). Increasing the level of these substances in water of Technology, Shahrood, Iran Full list of author information is available at the end of the article resources and wastewater led to increasing attention of © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Roudbari and Rezakazemi AMB Expr (2018) 8:91 Page 2 of 8 researchers and national and regional authorities to them elastic skin and reduction of organic chemical pollutants and several studies have been conducted to determine from liquid environments (Mahravan et al. 2016). their concentration in liquid environments. In a study in The aim of this study was to investigate the effect of Brazil in 2012, E1 and E2 concentrations in raw waste- ultrasound on the reduction of sewage hormones from water were determined 566 and 143 ng/L, respectively wastewater and not effluent. To achieve this purpose, (Pessoa et al. 2014). Also, in the study on wastewater samples were taken from the end of wastewater collec- treatment lagoons in the US in 2011, the concentration tion network before entering the first unit of the treat - of these hormones was reported 16.9 and 126 ng/L, ment plant. Investigations showed that very few studies respectively (Luo et al. 2017). Also, the study conducted have been done on the effects of ultrasound on reduc - in South Korea in 2004 indicated E1 hormone levels in ing hormones parameters such as the effect of power surface waters at 1 ng/L (Kim et al. 2007). Some of the and different frequencies, the effect of exposure time to studies on hormones are attributed to how to remove ultrasound, the effect of initial pH, and also the interac - or decrease the amount of them in water resources and tion of ultrasound frequency and power. Therefore, the wastewater. Different methods are used in these stud - aim of this study was to investigate the effect of power, ies; the most important methods are: activated sludge frequency, exposure time, initial pH, and also the inter- along with biological nutrient reduction (BNR) (Phil- action of ultrasound frequency and power on reduction lips et al. 2016) without reduction of biological nutrients efficiency of Estrone and 17 beta-Estradiol from munici - (nBNR) (Sornalingam et al. 2016), oxidation ditch (Li pal wastewater. Moreover, the main aspects of this tech- et al. 2013), aeration lagoon (Li and Ni 2011), the combi- nology have not been studied yet. nation of nanofiltration and reverse osmosis (Plósz et al. 2010), activated carbon adsorption (Foroutan et al. 2017; Materials and methods Furgal et al. 2015), peroxon process (Zhang et al. 2015), Chemicals water chlorination and photo-Fenton-like degradation All required chemicals including ferrous sulfate, sulfu- (Ifelebuegu et al. 2016). Wastewater treatment has been ric acid, and hydrogen peroxide were purchased from the subject of several recent studies (Baheri et al. 2014; Sigma-Aldrich. In this study, E1 and E2 hormones were Rezakazemi et al. 2018b; Muhammad et al. 2017; Rezaka- studied. The existence of these hormones in wastewater zemi 2018; Rezakazemi et al. 2011a, b, 2012, 2013, 2014; with higher concentrations than other hormones, as well Rezakazemi et al. 2018a, c; Shahverdi et al. 2013; Shira- as differences in chemical structure, molecular weight, zian et al. 2012). Indeed, the efficiency of these methods and their properties were the reasons for choosing these for reducing the hormones is different and some of them two hormones. The concentrations of these hormones in were unable to provide acceptable reduction efficacy wastewater were between 485 and 535 ng/L. Sulfuric acid (Wojnarowicz et al. 2014). According to studies, biologi- was used to adjust pH. Ferrous sulfate and hydrogen per- cal treatment methods and advanced treatment processes oxide were used for sample preparation in ECL method. have better ability to remove or reduce the amount of hormones. Although biological treatment can eliminate Wastewater characteristics and preliminary experiments hormones, some hormones remain in the effluent (Auriol Wastewater used in this study was prepared from Shah- et al. 2008). Advanced treatment methods have higher rood wastewater treatment plant. This treatment plant ability to remove hormone, but they are faced with two uses stabilization ponds process and is the largest treat- limitations of high costs and dangerous by-products for- ment plant in Semnan province. Samples were taken mation (Moreira et al. 2017). from the entry of the first pond. The characteristics of In general, most of the methods proposed for the wastewater are described in Table 1. Samples after col- removal of hormones are on a laboratory scale and there lecting were tested immediately to minimize biochemical are few real scale examples. One of the most effective changes. The amount of nitrogen ammonia and pH were methods to remove hormones without by-products gen- eration is ultrasound (waves with a frequency greater than 20 kHz). Ultrasound waves which had discovered Table 1 Characteristics of wastewater by Francis Galton in 1876 are produced by two meth- Parameter Value Unit Parameter Value UNIT ods: Piezoelectricity (interaction of mechanical pressure and electrical power), and Magnetostriction (Generation Total solids 1150 ± 65 mg/L TOC 156 ± 16 mg/L of ultrasonic waves in the electromagnetic field) (Mus - COD 340 ± 48 mg/L pH 7.2 ± 0.35 – ielak et al. 2016). This method is used for remedy of new BOD 225 ± 43 mg/L Ammonia nitro- 652 ± 34 mg/L gen injuries and old and chronic inflammations, recovery of E1 485 ± 32 ng/L E2 511 ± 16 ng/L Roudbari and Rezakazemi AMB Expr (2018) 8:91 Page 3 of 8 measured by C203 8 parameter test meter device (Hanna investigated. Also, each test was repeated 3 times, and Electronics Company) and benchtop pH meters (Cole- residual concentration of E1 and E2 hormones was meas- Parmer Co., Ltd Company). The pH meter was calibrated ured in the reactor effluent. before each use with pH 3, 7 and 10 buffer solutions. The biochemical oxygen demand (BOD) and chemical oxygen Analytical method demand (COD) measurements were determined follow- Hormone concentration was measured by electrolu- ing Standard Methods 5210 and 5220, respectively. Total minescence (ECL) method (Kische et al. 2016). For this solids (TS) were measured through evaporation in a fur- purpose, the joint product of Roche and Hitachi, Elec- nace at 105 °C for 1 h. sys 2010, was used. In this method, 35 μL of the sample with a specific estradiol antibody constitute an immune Reactor characteristics complex. Then the tiny spherical constituents coated In this study, a cylindrical reactor made of Plexiglas in with streptavidin are added to the environment that con- the amount of 1.5 L for the batch reactor was designed sequently, places of antibodies that are still empty can (Fig. 1). The reactor contents were stirred by a stirrer form an antibody mixture (Hapten) and are filled with it. magnet with low speed (450 rpm). The source of ultra - Then, all mixture transferred to a solid phase during an sound generation was the device Model UGMA-5000 interaction and being pulled into measuring cell. In this (Sonotek Company) with three transducers 30, 45 and cell, micron particles magnetically trapped on the elec- 60 kHz equipped with a titanium probe with a diam- trode surface and substances that have not been trapped eter of 20 mm, operated over a frequency range of will be removed by Procell. Then voltage is added to the 25–250 kHz with a display resolution of 0.01 Watts and electrode to cause emission of chemiluminescence light. ± 4% accuracy leading for increased repeatability. The The amount of the light is measured by a light multiplier. probe submerging depth in the reactor was 22.5 cm (half Then, the result is taken to the calibration curve and the of the reactor depth). amount of hormones was determined (Cacho et al. 2016). A detection limit of ECL was adjusted to 1–500 ng/L. Experimental setup In this study, the effect of ultrasound power, frequency Statistical analysis and exposure time and also the initial pH of waste- In this study, the effects of frequency, power, exposure water on the reduction of E1 and E2 hormones were time, and initial pH were evaluated on the amount of investigated. For this purpose, the effect of powers (70 the E1 and E2 reduction from municipal wastewater. and 110 W), frequency (30, 45 and 60 kHz), exposure SPSS version 19 was used for statistical analysis. One time (30, 60, 90 and 120 min) and pH (3, 7 and 10) were way ANOVA was used for evaluating the effect of fre - quency, exposure time and pH and Independent Sample T Test was used for evaluating the effect of power. Tukey HSD was used to determine which value of the variable was effective to remove hormones. Univariate analysis was used to evaluate the effect of interaction and Levene test is used to determine the equal or unequal variances in different groups for each variable. When the variances in different groups were not being equal, Kruskal–Wallis test (its nonparametric equivalent) and Mann–Whitney test were used instead of one-way ANOVA and inde- pendent sample T-Test, respectively. Results Table 1 shows the wastewater characteristics which were used in the study. As can be seen, the wastewater was clearly representative of municipal wastewaters. The studies performed by Sun et al. (2016) and Renuka et al. (2016) showed similar results. The results showed that ultrasound waves had the high ability on reducing hormones E1 and E2 (Tables 2 and 3). Figure 2 shows the effect of power on E1 and E2 reduction. As can be Fig. 1 Schematic of reactor seen, with increasing the ultrasound power increases Roudbari and Rezakazemi AMB Expr (2018) 8:91 Page 4 of 8 Table 2 Reduction rate (%) of estrogen by ultrasound at different time, power, pH and frequency (Mean ± SD) pH Power (W) Frequency (KHz) Time (min) 30 60 90 120 3 70 30 12.4 ± 2.3 12.7 ± 1.4 13.1 ± 1.3 13.2 ± 1.3 3 70 45 13.3 ± 1.8 13.5 ± 1.6 13.8 ± 1.2 14.5 ± 1.2 3 70 60 14.3 ± 1.3 15.6 ± 1.4 15.8 ± 1.2 16.5 ± 1.3 3 110 30 20.5 ± 2.4 21.1 ± 2.1 21.5 ± 1.7 21.9 ± 1.6 3 110 45 22.3 ± 1.5 22.7 ± 1.5 22.9 ± 1.4 23.3 ± 1.1 3 110 60 23.5 ± 1.2 23.7 ± 1.2 24.1 ± 1.4 24.3 ± 1.2 7 70 30 26.3 ± 1.4 26.7 ± 1.2 29.6 ± 1.5 31.2 ± 1.4 7 70 45 32.5 ± 1.2 32.3 ± 1.1 33.8 ± 1.3 35.2 ± 1.6 7 70 60 31.5 ± 1.3 33.1 ± 1.2 35.6 ± 1.2 38.9 ± 1.8 7 110 30 39.1 ± 2.5 40.5 ± 2.9 40.8 ± 2.3 42.3 ± 3.1 7 110 45 42.1 ± 3.2 43.5 ± 2.7 44.9 ± 2.6 46.1 ± 2.5 7 110 60 46.0 ± 2.5 46.1 ± 2.8 47.8 ± 2.6 49.2 ± 2.5 10 70 30 67.1 ± 3.1 68.9 ± 3.6 69.5 ± 2.7 70.6 ± 2.8 10 70 45 70.8 ± 2.6 71.5 ± 2.5 74.2 ± 2.7 75.3 ± 2.6 10 70 60 75.6 ± 2.5 75.9 ± 2.6 77.5 ± 2.8 79.6 ± 2.6 10 110 30 81.3 ± 2.6 81.9 ± 2.5 82.6 ± 3.1 84.6 ± 3.1 10 110 45 85.8 ± 3.7 86.9 ± 2.6 87.2 ± 3.5 90.9 ± 3.2 10 110 60 91.1 ± 3.1 91.2 ± 3.1 92.2 ± 3.2 94.2 ± 2.7 Table 3 Reduction rate (%) of 17 beta-estradiol by ultrasound at different time, power, pH and frequency (Mean ± SD) pH Power (W) Frequency (KHz) Time (min) 30 60 90 120 3 70 30 12.2 ± 1.8 12.5 ± 1.6 13.0 ± 1.1 13.1 ± 1.3 3 70 45 13.1 ± 1.2 13.2 ± 1.3 13.3 ± 1.2 13.7 ± 1.4 3 70 60 13.8 ± 1.1 14.5 ± 1.2 15.1 ± 1.2 15.8 ± 1.1 3 110 30 19.8 ± 1.3 21.0 ± 1.3 21.2 ± 1.2 21.6 ± 1.4 3 110 45 21.9 ± 1.3 22.3 ± 1.2 22.5 ± 1.4 23.1 ± 1.4 3 110 60 23.1 ± 1.4 23.5 ± 1.4 24.0 ± 1.3 24.1 ± 1.4 7 70 30 25.8 ± 1.6 26.4 ± 1.5 28.8 ± 1.5 30.8 ± 1.3 7 70 45 31.8 ± 1.2 32.1 ± 1.3 32.5 ± 1.2 34.5 ± 1.2 7 70 60 30.8 ± 1.3 32.5 ± 1.2 34.2 ± 1.4 37.5 ± 1.6 7 110 30 38.6 ± 1.3 39.8 ± 1.2 40.5 ± 1.2 41.8 ± 1.4 7 110 45 41.8 ± 1.3 42.9 ± 1.4 44.5 ± 1.3 45.8 ± 1.4 7 110 60 43.0 ± 1.2 45.6 ± 1.4 46.2 ± 1.3 48.2 ± 1.5 10 70 30 66.5 ± 2.3 67.8 ± 2.1 69.2 ± 2.2 70.8 ± 2.3 10 70 45 70.9 ± 2.4 72.3 ± 2.2 74.2 ± 2.3 75.5 ± 2.1 10 70 60 75.3 ± 2.2 75.9 ± 2.1 76.8 ± 2.3 78.5 ± 2.1 10 110 30 81.9 ± 2.4 82.3 ± 2.3 82.5 ± 2.8 83.9 ± 2.6 10 110 45 86.2 ± 2.5 86.9 ± 2.6 86.6 ± 2.8 88.9 ± 2.6 10 110 60 90.9 ± 2.4 91.3 ± 2.5 91.8 ± 2.3 93.6 ± 2.2 the efficacy reduction of E1 and E2. Figure 3 shows the the effect of exposure time on E1 and E2 reduction. As effect of frequency on E1 and E2 reduction. As can be can be seen, with increasing exposure time, the reduction seen, with increasing ultrasound frequency, the reduc- efficacy of E1 and E2 somewhat has increased but the not tion efficacy of E1 and E2 has increased. Figure 4 shows significant. Figure 5 shows the effect of pH on E1 and E2 Roudbari and Rezakazemi AMB Expr (2018) 8:91 Page 5 of 8 Fig. 2 Eec ff t of power on E1 and E2 reduction (frequency = 45 kHz, pH 7) Fig. 5 Eec ff t of pH on E1 and E2 reduction (power = 110, frequency = 60 kHz) of E1 and E2 after 45 min while other parameters changes in the range of their operations and ultrasound frequency and power had a significant effect on reduction efficiency but exposure time had no significant effect. Also despite the fact that the power and frequency individually had a significant effect on hormones reduction, but their con - current effect was reductive. With the increasing initial pH, reduction efficacy increased due to the increased production of hydroxyl radicals. The result indicated that the ultrasound has high ability to remove Steroid Fig. 3 Eec ff t of frequency on E1 and E2 reduction (power = 70, pH 7) hormones from municipal wastewater, so due to non- generation of dangerous by-products and low electricity requirement; this method can be considered as a valuable strategy for reduction or destruction of hormones from water resources. The effect of frequency and power on reducing E1 and E2 was significant but the effect of exposure time was not significant. Statistical analysis of Univariate showed that the power individually had effective rates of 89.9 and 90.1% on E1 and E2 reduction, respectively, and the fre- quency individually had effective rates of 80.1 and 82.2% on E1 and E2 reduction, respectively. But the concur- rent effect of frequency and power was significant (P value for E1 and E2 were 0.008 and 0.006, respectively) and decreases (reduction efficacy for E1 and E2 were 68.5 and Fig. 4 Eec ff t of exposure time on E1 and E2 reduction (power = 110, 70.1%, respectively). frequency = 60 kHz) In fact, ultrasound waves lead to extensive destruc- tion of organic materials, particularly macromolecules and massive organic materials by the formation of reduction. As can be seen, with increasing pH, the reduc- hydroxyl radical and cavitations phenomenon (Budi- tion efficacy of E1 and E2 has increased. man and Wu 2016; Wu et al. 2012). E1 and E2 hormones have strong oxidizing properties, so they are easily oxi- Discussion dized by hydroxyl free radicals produced by ultrasound In this study, the effect of power, frequency, exposure (Renuka et al. 2016). Also, these compounds are exposed time, initial pH, and also the concurrent effect of power to cavitation (Hotspot) made by ultrasound and they will and frequency on Estrone and 17 beta-Estradiol reduc- remove because they have carbon ring of cyclohexane tion from municipal wastewater were investigated. and cyclopentane types and desirable volatility prop- According to the results, ultrasound can reduce 85–96% erties (Jia et al. 2015). Andaluri et al. (2012) in a study Roudbari and Rezakazemi AMB Expr (2018) 8:91 Page 6 of 8 Authors’ contributions showed that ultrasound can destruct some hormones The work is a product of the intellectual environment of the whole team; and through the production of hydroxyl radical and cavita- that all members have contributed in various degrees to the analytical meth- tion phenomenon. ods used, to the research concept, and to the experiment design. All authors read and approved the final manuscript. According to Sono-chemical theory, when the ultra- sound power increases more than the cavitations thresh- Author details old, bubbles are formed faster and more and release Center for Social and Behavioral Sciences Research, Shahroud University of Medical Sciences, Shahroud, Iran. Faculty of Chemical and Materials Engi- its energy easily (Andaluri et al. 2012), so a large num- neering, Shahrood University of Technology, Shahrood, Iran. ber of cavitations bubbles explode with high energy in a short time and destroy hormonal molecules attached Acknowledgements Not applicable. to themselves, thus the efficiency of hormones reduc - tion increases. Statistical analysis of independent sam- Competing interests ple T-Test showed that there is a significant difference The authors declare that they have no competing interests. between E1 and E2 concentrations in different powers in Availability of data and materials reactor influent and effluent (P = 0.001 for both hor- value The datasets generated and/or analysed during the current study are available mones). The results of the study are consistent with the from the corresponding author on reasonable request. study of Andaluri et al. (2012). They studied ultrasonic Consent for publication oxidation of diethyl phthalate and concluded that with Not applicable. increasing the power, the removal efficacy increases. Ethics approval and consent to participate The increasing of frequency leads to increasing the Not applicable. number of cavitations bubbles and also the number of hydroxyl radicals in the environment (Suri et al. 2007), so Funding Not applicable. the reduction efficacy of E1 and E2 increases. The statisti - cal analysis of one way ANOVA showed that there is a Publisher’s Note significant difference between E1 and E2 concentrations Springer Nature remains neutral with regard to jurisdictional claims in pub- in different frequencies in reactor influent and effluent lished maps and institutional affiliations. (P for E1 and E2 are 0.001 and 0.002 respectively). value Received: 5 April 2018 Accepted: 23 May 2018 Also, statistical analysis of Tukey showed there is a sig- nificant difference between 30 and 60 kHz frequencies (P for E1 and E2 are 0.001 and 0.002, respectively) value and between 45 and 60 kHz frequencies (P for E1 and value References E2 are 0.002 and 0.001, respectively) on E1 and E2 reduc- Aker AM, Watkins DJ, Johns LE, Ferguson KK, Soldin OP, Del Toro LVA, Alsha- tion. Suri et al. (2007) confirmed the effect of frequency wabkeh AN, Cordero JF, Meeker JD (2016) Phenols and parabens in rela- tion to reproductive and thyroid hormones in pregnant women. Environ on hormones reduction in their study. Res 151:30–37 In fact, because the formation of hydroxyl radical Andaluri G, Rokhina EV, Suri RPS (2012) Evaluation of relative importance of and cavitations bubbles begin to operate in very short ultrasound reactor parameters for the removal of estrogen hormones in water. 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Published: Jun 1, 2018
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