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Chapter 4: Pilot study
Assessment of the target chemicals in the YES and T47D-KBluc bioassays
The target chemicals were assessed in the YES and T47D-KBluc bioassays. DINP was not one of the initially selected target chemicals for this project, but was included after it was detected in many of the samples. BPA, NP, DBP, E2, E1 and EE2 showed estrogenic activity in the YES and T47D-KBluc bioassays and DEHP only in the T47D-KBluc bioassay. DEHA and DINP did not show estrogenic activity at the tested concentrations and none of the chemicals showed anti-estrogenic activity. All the target chemicals showed estrogenic responses at much lower concentrations in the T47D-KBluc bioassay compared to the YES bioassay, making the T47D-KBluc bioassay a much more sensitive bioassay. BPA, NP, DBP and DEHP were cytotoxic at higher concentrations. Graphs of the estrogenic responses of the target chemicals in the YES and T47D-KBluc bioassays are shown in Figure 4.1 and Figure 4.2 respectively.
DBP and DEHP were not able to reach the maximum estrogenic response obtained by E2. DBP only reached 5% of the maximum E2 response in the YES and 12% in the T47D-KBluc bioassay. DEHP reached 21% of the maximum E2 response before cytotoxicity was observed. Chemicals do not always react the same in different in vitro bioassays. Although it was not observed in this study, DEHP showed anti-estrogenic activity in MVLN cells.188 A different study reported weak estrogenic activity for DINP in a YES screen.211 Similar to this study, weak estrogenic activity was observed for DBP in MVLN188 and CV-1 cells.189 The relative potencies obtained in this study are similar to relative potencies reported in the ERα CALUX bioassay (EE2: 1.86; BPA: 2.5E-05; NP: 4.6E-05), except for E1 (0.02) that showed a higher relative potency in the T47D-KBluc cells.105
Validation of extraction method
In order to evaluate the suitability of the extraction method recommended by Oasis221, it was compared to the method described in the WRC toolbox222 and a method used by Leusch et al.210 The three different extraction methods are compared in Table 4.2.
Samples were prepared by spiking 1 L of ddH2O with 0; 2 or 20 ng/L E2. The three extraction methods were carried out using glass and disposable (plastic) pipettes for comparison. The re-use of glass pipettes could result in the contamination of samples if the pipettes are not cleaned properly and disposable pipettes might contaminate samples if plastic constituents leach from the pipettes into the samples.
Extracts were analysed for estrogenic activity using the YES and T47D-KBluc reporter gene bioassay and were also analysed for the target chemicals using UPLC-MS. The results obtained in the bioassays and the UPLC-MS results for E2 and BPA are summarised in Table 4.3. Low levels of BPA were detected in all of the samples, but this was attributed to contamination of one of the solvents used in the preparation of the samples, as the solvent and extraction controls also tested positive for BPA. NP, DEHA, DBP, DEHP, E1 and EE2 were not detected.
The low recovery seen for E2 using UPLC-MS could be explained by the fact that the E2 used to spike the samples (Sigma cat. no. E8875), were not from the same supplier as the E2 (Fluka cat. no. 75262) used for the calibration curves for the analysis of the samples. Beresford et al.232 also reported different results when testing the same chemical from different batches and suppliers (when testing estradiol-3-sulfate in the YES bioassay). They’ve suggested that the different results could be ascribed to a difference in the purity or composition of the chemicals and that storage conditions could also have had an influence.
Oasis HLB cartridges are efficient to retain a wide range of structurally diverse pollutants, as the cartridges exhibit both hydrophilic and lipophilic retention characteristics.233 The results confirmed that the Oasis extraction method was suitable for the purposes of this project. No clear evidence of leaching from the disposable pipettes could be seen. It was therefore decided to use new glass pipettes for the solvents (one pipette dedicated to each solvent) and disposable pipettes for loading the samples onto the cartridges.
Recoveries of target chemicals using UPLC-MS
In order to determine the recoveries of the target chemicals after the extraction process, triplicate 1L ddH2O samples were spiked with a standard cocktail containing all the target chemicals. The final concentration of each target chemical was 200 ng/L. The spiked and unspiked (control) samples were extracted and analysed for the target chemicals using UPLC-MS. The recoveries are tabulated in Table 4.4.
Recoveries above 100% indicate background levels of the analytes in laboratory water or solvents used for UPLC-MS. Background levels of DBP and DEHA were especially high. However DBP was not detected in any of the extraction control samples or solvents. DEHA was not detected in any of the solvents but was detected in one of the nine extraction control samples.
Phase 1 – Distribution point water
Bioassays for estrogenic activity
None of the samples from the distribution points were above the dl in the YES bioassay. The results obtained with the T47D-KBluc bioassay are tabulated in Table 5.1 (Pretoria) and Table 5.2 (Cape Town).
Six of the distribution points in Pretoria and seven of the distribution points in Cape Town showed estrogenic activity in the T47D-KBluc bioassay for at least one sampling period. Estrogenic activity was detected in all four sampling periods in PTA01, CPT04, CPT06 and CPT08. The sampling period with the highest number of positive samples was July 2014 (winter) for Pretoria and Cape Town. The EEq values ranged from below the dl to 0.089 ng/L in Pretoria and from below the dl to 0.114 ng/L in Cape Town. More samples tested positive for estrogenic activity in the Cape Town distribution points (53%) compared to the Pretoria distribution points (30%), but in general the Pretoria samples had higher estrogenic activities (average EEq = 0.014 ng/L) compared to Cape Town samples (average EEq = 0.008 ng/L). None of the samples showed anti-estrogenic activity.
Target chemical analyses
Although DINP was not one of the selected target chemicals, it was detected in many of the samples during UPLC-MS analysis and it was therefore decided to include DINP in the project.
The dl and quantification limit (ql) obtained for each target chemical using the UPLC-MS method are summarised in Table 5.3. Due to the fact that the samples were concentrated a 1000 times, target chemicals could be detected at concentrations a 1000 times lower than the dl. For example, for BPA the dl is 0.5 ng/L and ql is 5 ng/L, but because the samples were concentrated 1000 times, BPA could be detected at 0.0005 ng/L and quantified from 0.005 ng/L.
Phase 2 – Bottled water
Mineral composition
The mineral composition (as it was printed on the labels of the bottles) of the ten brands of bottled water selected for this study is summarised in Table 5.19. The pH of the different brands of water varied from 4.5 to 8.
Bioassays for estrogenic activity
None of the samples were above the dl in the YES bioassay and no cytotoxicity was observed.
In the T47D-KBluc bioassay, eight samples had estrogenic activity, with EEq values ranging from below the dl to 0.011 ng/L (Table 5.20). Only one sample incubated at 20°C were above the dl of the bioassay, the other positive samples were all incubated at 40°C (four in dark and three in light conditions). The highest EEq (0.011 ng/L) were BTW05, incubated at 40°C in the dark. None of the samples had anti-estrogenic activity or cytotoxicity.
Declaration
Summary*
Acknowledgements
Table of Contents
List of Figures
List of Tables
List of Abbreviations
Chapter 1: Literature Review
1.1. The endocrine system
1.2. Endocrine disrupting chemicals (EDCs)
1.2.1. Definition of EDCs
1.2.2. Classes of EDCs
1.2.3. Routes of exposure
1.2.4. Health effects associated with EDC exposure
1.2.5. EDC activity
1.2.5.1. How do EDCs disturb the hormonal system?
1.2.5.2. The EDC dose response curve
1.2.5.3. The mixture effect
1.2.5.4. Exposure to EDCs during critical windows .
1.2.6.6. Multigenerational and transgenerational effects
1.3. EDCs in the aquatic environment
1.3.1. Natural and synthetic steroids in sewage effluent .
1.3.2. Pharmaceuticals and hospital effluents
1.3.3. Personal care products
1.3.4. Household products and industrial effluents
1.3.5. Agricultural effluents
1.4. Removal of EDCs through water treatment processes
1.4.1. Removal of EDCs from wastewater
1.4.2. EDCs in drinking/tap water
1.5. Bottled water as an alternative to tap water
1.5.1. Sources of contamination of bottled wate
1.5.1.1. Endocrine activity
1.5.1.2. Bisphenol A (BPA)
1.5.1.3. Nonylphenol (NP)
1.5.1.4. Phthalates
1.5.1.5. Di(2-ethylhexyl) adipate (DEHA)
1.5.2. Factors influencing the migration of chemicals from bottles into water content
1.6. Testing methodologies for EDCs
1.6.1. Sample preparation
1.6.2. Bioassays and non-cellular in vitro assays
1.6.3. Whole organism assays
1.6.4. Chemical analysis
1.7. Health risk assessment of EDCs
1.7.1. Hazard identification
1.7.2. Dose response assessment
1.7.3. Exposure assessment
1.7.4. Risk characterization
Chapter 2: Aims and objectives of the study
2.1. Hypothesis
2.2. Aims and objectives
2.2.1. Phase 1 – Distribution point water
2.2.2. Phase 2 – Bottled water
2.2.3. Phase 3 – Health risk assessment
Chapter 3: Materials and Methods
3.1. General laboratory procedures
3.2. Phase 1 – Distribution point water
3.3. Phase 2 – Bottled water
3.4. Phase 3 – Health Risk Assessment
3.5. Ethical considerations
Chapter 4: Pilot study
4.1. Assessment of the target chemicals in the YES and T47D-KBluc bioassays
4.2. Validation of extraction method
4.3. Recoveries of target chemicals using UPLC-MS
Chapter 5: Results
5.2. Phase 1 – Distribution point water
5.3. Phase 2 – Bottled water
5.4. Comparison between distribution point and bottled water
5.5. Phase 3 – Health Risk Assessment
Chapter 6: Discussion and Conclusions
6.1. General discussion on methods
6.2. Distribution point water
6.3. Bottled water
6.4. Comparison between distribution point and bottled water
6.5. Health risk assessment
6.6. Environmental and Public Health considerations
6.7. Conclusions
6.8. Recommendations
References
Appendix
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