1. Using ultrasonic liquid extraction for estrogens analysis in sludge by HPLC with fluorescence detection
Vitória Lourosa,
Diana Limab, Jorge Leitãoc, Valdemar Estevesb, Helena Nadaisa 
a Department of Environment and Planning, CESAM, University of Aveiro, Aveiro
b Department of Chemistry, CESAM, University of Aveiro, Aveiro
c Centre for Biological and Chemical Engineering, IBB, Instituto Superior Técnico, Lisbon
Abstract
A method for the simultaneous determination of estrone (E1) and 17α-ethinylestradiol (EE2) in fresh digested sludge samples was developed, based on ultrasonic liquid extraction (ULE) and high-performance liquid chromatography with fluorescence detector (HPLC-FLD). The developed method is fast, cheap and easy-to-use.
During the pretreatment procedure the standards were spiked onto the fresh sludge and extracted using three-steps extraction procedure with methanol and acetone as extraction solvents. Good calibration linearity, recovery and precision, for E1 and EE2, in real digested sludge samples were observed. The recoveries of E1 and EE2 in digested sludge were   0.9% and 104  4%, respectively. Limits of detection (LOD) for E1 and EE2 in fresh sludge were detected up to 67.5 µg L−1 and 7.0 µg L−1, respectively. In solid phase of sludge LODs of 1.2 µg g−1 for E1 and 0.1 µg g−1 for EE2 were achieved.
Introduction
Recently, a group of organic pollutants called endocrine-disrupting chemicals (EDCs) has attracted some attention due to their impact in the environment and living beings. Natural estrogens estrone (E1), 17β-estradiol (E2) and the synthetic estrogen 17α-ethinylestradiol (EE2) are reported as the EDCs with higher disrupting potency, even at trace levels. Wastewater Treatment Plants (WWTPs) are a significant point source of EDCs released into the environment. In the literature is reported that E2 is readily oxidized to E1 during the sewage treatment process. Due the high sorption potential of estrogens, in WWTPs, large proportion of removed estrogens are adsorbed onto sludge. Therefore, it is crucial to develop a selective, reliable and affordable analytic method for the simultaneous determination of E1 and EE2 in fresh digested sludge from WWTP.
Experimental procedure
The treatment of digested sludge sample is illustrated in Fig 1.
Results
The extraction recovery rates were determined by spiking a known amount of E1 and EE2 into fresh digested sludge with contact times of 5 min, 30 min and 1 h. This parameter was calculated by the estrogen mass measured in fresh sludge after spiking with estrogen divided by total mass spiked of each compound in sample. The individual mass measured of E1 and EE2 was determined by adding the masses quantified in liquid and in solid phases.
Results indicated good recovery efficiencies of the overall method, which ranged from % to % for E1 and EE2, respectively, indicating good accuracy; also good precision were observed with relative standard deviation <4% (Table 1). A comparison between the methodology applied in this study and in literature is presented in Fig 2.
The different adsorption values obtained for E1 and EE2 (Fig 3) may be associated with the higher sorption potential of EE2. At contact times of 5 min, 30 min and 1h, no differences were observed of mass E1 and EE2 in liquid phase and solid phase.
Table 1 / E1 and EE2 analytical curves and recovery results.
Conclusion
Comparing with the other methods reported in the literature, ULE-HPLC-FLD presents the following advantages: high extraction recoveries, rapid and easy-to-use analysis. Overall, the method suggested in this study shows good efficiency for determination of E1 and EE2 in fresh sludge matrix.
References
[1] Ternes, T.A., Andersen, H., Gilberg, D., Bonerz, M. (2002) Determination of Estrogens in Sludge and Sediments by Liquid Extraction and GC/MS/MS, Anal. Chem., Vol. 74, pp. 3498–3504.
[2] Nie, Y., Qiang, Z., Zhang, H., Adams, C. (2009) Determination of endocrine-disrupting chemicals in the liquid and solid phases of activated sludge by solid phase extraction and gas chromatography-mass spectrometry, J. Chromatogr. A, Vol
Acknowledgements
Thanks are due for the financial support to CESAM (UID/AMB/ POCI FEDER ), to FCT/MEC through national funds, and the co-funding by the FEDER, within the PT2020 Partnership Agreement and Compete Vitoria Louros and Diana Lima would like to thank for their doctoral (SFRH/BD/112907/2015) and post-doctoral (SFRH/BPD/80315/2011) grants, respectively.
Analyte
Linear range
Correlation coefficient
Linearity
Limit of detection
Extraction recovery a
Fresh sludge
Solid phase of sludge
(μg L-1)
(r)
(%)
(μg g-1) E1
97.164
67.5
1.2
102.9  0.9 b
EE2
10-500
99.451
7.0
0.1
104  4 b
a Values obtained for a mass spike of 100 μg E1 and 1 μg EE2 with contact times of spiked estrogen with fresh sludge of 5 min, 30 min and 1 h.
b Mean value  standard deviation (n = 9).
Our study
Literature
Standards spiked onto freeze-dried sludge [1, 2]  overestimation of recovery rates of this compounds and low LOD
Standards spiked onto fresh sludge
Fresh digested sludge samples (50 mL)
Longer consumption times  degradation of estrogens during cleanup steps [1]
Spike
Addition of 100 μg E1 and
1 μg EE2 with contact times of 5 min, 30 min and 1 h
Fig 2 / Comparison between the methodology applied in this study and in literature.
Centrifugation
(4000 rpm, 10 min)
Solid phase
Liquid phase
Filtration (Pore size 0.2 μm)
Storage (-80 ºC)
More than 78.7% of spiked E1 and 90.7% of spiked EE2 were adsorbed onto sludge phase after 5 min of contact time
Storage (4 ºC)
Freeze-dried
3  extraction
Shaken, 1 min
ULE*, 1h
Centrifugation (4000 rpm), 10 min
Filtration (Pore size 0.2 μm)
Analysis by HPLC-FLD
Fig 1 / Schematic diagram for analysis of hormones in liquid and solid phases of digested sludge. *Ultrasonic liquid extraction (ULE) was carried out with 9 mL of methanol, 4.5 mL of methanol and 4.5 mL of acetone per gram of dried sludge.
Fig 3 / Mass profiles of E1 (a) and EE2 (b) using the spike of 100 μg E1 and 1 μg EE2 in fresh digested sludge under anaerobic condition.