Passive in-situ estrogenic potency sampling with DGT-YES
The purpose of this research was to investigate the presence of bisphenolic plastics and synthetic estrogens in municipal water systems, as well as along the Waikato River. The concentration of these compounds was assessed using a novel combination of methods that allowed the actual levels of endocrine disruption to be ascertained. This was based on diffusive gradients in thin films (DGTs) coupled with high-performance liquid chromatography/mass spectrometry analysis (HPLC/MS) and the Yeast Estrogen Screen (YES). DGT estimated time-integrated concentrations while HPLC/MS ensured a robust, automated and sensitive routine analysis. Most significantly, YES, a biomarker-based assay, was deployed to quantify the total estrogenic potency of the compounds adsorbed to the DGT matrix. The unique dimension of this research was to extend the application of DGTs to estrogen sampling and to combine the DGT and YES technique to provide in situ quantification of the estrogenic potency of water bodies through time. DGTs were developed specifically for the sampling of BPA, BPAF, E2 and EE2. Several physical-chemical and chemical analyses were conducted to attest the suitability of the agarose (1.5%) and agarose (1.5%) – activated charcoal (1%) chosen respectively as diffusive gel and binding gel. They were found to be suitable for the detection of organic molecules with a bigger steric hindrance and agarose (1.5%) – AC (1%) appeared suitable for long deployments thanks to its high thermal resistance. An HPLC-MS method was optimised to quantify the compounds of interest. A methanol (MeOH, NH4OH 0.06 M)/water (H2O, NH4OH 0.06 M) fast gradient elution program of 15 min was chosen to elute the analytes, MS parameters were optimized for each compound and the negative mode was selected to perform the fragmentation. LODs and LOQs of all targets resulted in the nanomolar range assuming 24 h as deployment time at 25°C with a 0.54 mm thick diffusive gel. The sensitivity of the method was increased in the sub-nanomolar range by adopting long deployment times (18 days) thus enhancing the accumulation of the targets.The YES was successfully developed to quantify the total estrogenic potency of the DGTs eluted. The dose-response calibration curve of E2, employed as a control, demonstrated LOD and experimental EC50 values in the sub-nanomolar range. EE2, BPA and BPAF demonstrated agonistic endocrine activity, in particular EE2 being more potent than E2, while BPAF and BPA were less potent. The environmental monitoring assessed the efficiency of removal of the selected ECs during the water treatment processes from river-to-tap and effluent-to-treated wastewater in Hamilton as well the water quality from source-to-outfall of the Waikato River. The HPLC-MS and YES results highlighted that primary treatments were not suitable for the removal of bisphenolic plastics and estrogens both at drinkable and wastewater treatment plants in Hamilton where they appear to worsen the water quality making these pollutants more available. At both treatment plants, the concentration and the estrogenic activity of the targets appeared to fluctuate around the same value, pointing out the inefficiency of removal of these compounds with the actual treatments. EE2 contributed the most to the estrogenic activity due to its higher concentration and higher potency at all sites. The concentration and the estrogenic potency of all targets were found to be higher downstream at all sites monitored along the Waikato River. EE2 was the compound with the highest concentration at all sites. A moderate worsening of water quality was apparent moving from Taupo to Tuakau. The most polluted sites were Hamilton and Huntley after the outlets of wastewater treatment plants that serve the main conurbations in the area. The EDC concentrations recorded in November 2017 and February 2018 during the monitoring of the Waikato River at Hamilton upstream and downstream sites showed a significant dilution due to the weather conditions. The data from a previous monitoring of the Waikato River adopting an SPE-HRGC/MS method were compared with those of the DGT-HPLC/MS investigation. The water quality appeared slightly worsened at all sites from 2013 to 2018 due to increased concentrations of BPA and EE2. The DGT-HPLC/MS quantification with the YES assay proved that not only active samplings but passive samplings as well can be coupled with a bioassay. This combined approach had the excellent potential to provide a reliable total estrogenic potency evaluation with time integrated concentrations. The use of the assay allowed estimation of the total estrogenic potency of the eluted DGT. The eluate appeared enriched by other compounds with estrogenic activity, suggesting this type of DGT could be suitable for the sampling and accumulation of other analytes that could be further investigated via HPLC/MS analysis. Compared to the traditional active approaches, DGT ensured sampling and concentration in one step, was easy to prepare and to use, required a very simple sample preparation step prior to analysis, and provided sensitivity in the sub-nanomolar range comparable to active sampling analyses. The YES assay was responsive, robust and easy to use compared to other assays that require laborious procedures and longer times for estrogenic potency evaluation and high sensitivity in the sub-nanomolar range comparable to other assays.
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