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Results generated by several Water Research Commission studies

Hormone and hormone mimicking activity in sewage effluent, surface water and treated drinking water Laetitia Slabbert. Results generated by several Water Research Commission studies Various institutions participated in the studies:

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Results generated by several Water Research Commission studies

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  1. Hormone and hormone mimicking activity in sewage effluent, surface water and treated drinking waterLaetitia Slabbert

  2. Results generated by several Water Research Commission studies Various institutions participated in the studies: CSIR, University of Stellenbosch, AMPATH, University of Pretoria, Environment Canada, Agricultural Research Council, SABS

  3. Outline • Background: endocrine disruption • Study area and sampling sites • Sample preparation • Detection methodologies • Results • Conclusions

  4. BackgroundHormone (endocrine) disruption – effect of natural and man-made chemicals on the endocrine systems of wildlife and man

  5. Wildlife: reproductive abnormalities; decreased fertility; impaired thyroid and immune system functionHumans: Effects on prenatal neurological development, neuro-endocrine and immune system function; decreased fertility; prevalence of testicular, uterine and breast cancer

  6. Natural EDCs: phyto- and mycoestrogens; human estrogens (17β-estradiol) and metabolites (estriol, estrone)

  7. Man-made EDCs

  8. EDCs enter the water environment via effluent discharge and run-off

  9. Sewage treatment plants - important sources of natural and synthetic estrogen contamination of aquatic environment • Pregnant females: 30 mg estrogen per day • Conjugates of sulphuric and glucuronic acids • Bacteria (E. coli) can catabolise metabolites to free steroids • Faeces contains large numbers of E. coli

  10. Study area

  11. Sewage treatment plants

  12. Effluent samples • 24 h composite samples • 5 consecutive days • Before chlorination

  13. Surface water • Hartebeespoort Dam • Rietvlei Dam • Vaal River Grab samples on four occasions

  14. Drinking water treatment plants

  15. Water samples • Grab samples • Four sampling occasions

  16. Sample preparation In vitro biological assays and estrogen analysis – solid phase extraction, residue dissolved in organic solvent Liquid-liquid extraction used for residue analysis In vivo test – effluent sample

  17. Detection methodologies • Biological tests: • In vitro tests • Recombinant yeast screen (YES) • Estrogen receptor-mediated chemical activated luciferase gene expression assay (ER-CALUX) • Primary rainbow trout hepatocyte (PRTH) test • In vivo test • 8- and 14-day zebrafish exposure • Chemical analysis: • GC-MS

  18. YES • Human estrogen receptor • Binding to receptor induces expression of the lac-Z gene carried by expression plasmids • Leads to activation of ß-galactosidase • Metabolism of yellow chlorophenol red-ß-D-galactopyranosid (CPRG) to a red product – assessed with a micro-spectrophotometer

  19. 17ß-estradiol 3 days 7 days 10 days Incubation time

  20. YES 17β-estradiol EC50’s 7.15-22.07 ng/ℓ

  21. ER CALUX assay • Human breast cancer cells • Exposure to estrogens - binding to endogenous ER • Leads to induction of luciferase gene • Assayed by lysing cells, adding substrate luciferin and measuring light output

  22. PRTH test - Estrogens bind with estrogen receptor in cells, which will trigger vitellogenin (VTG) production. VTG is a precursor of egg yolk - VTG will accumulate in the extracellular medium in 96-well microplates - Measured indirectly via alkali-labile phosphate groups using a micro-spectrophotometer

  23. Zebrafish test Sewage samples • Adult male fish exposed in aquaria for 8 days • Blood collected from tail vein, centrifuged and plasma used for VTG ELISA (enzyme linked immuno sorbent assay) in 96-well microplates • Histological analysis on gonads to determine sex Water samples • Juvenile fish exposed for 14 days • Homogenates used for VTG ELISA in 96-well microplates

  24. Female specimen - Male testicular tissue vitellogenic follicels

  25. YES results

  26. Sewage effluent

  27. River and dam water

  28. Final drinking water

  29. ER-CALUX test results

  30. Sewage effluent

  31. River and dam water

  32. Final drinking water

  33. Comparison of in vitro test results with values reported in literature Sewage effluent • 0.2-25 ng EEQ/ℓ E-screen – Germany • <16 ng EEQ/ℓ YES and ER-CALUX – Netherlands • <1.0-147 ng EEQ/ℓ YES – USA • 4-35 ng EEQ/ℓ modified YES – Japan • As high as 23.5 ng EEQ/ℓ YES – Study by Arijs on points of discharge in rivers/streams in the Pretoria-Johannesburg-Vereeniging area

  34. Comparison of in vitro test results with values reported in literature Surface water

  35. PRTH test results

  36. Sewage effluent

  37. River and dam water

  38. Final drinking water

  39. Zebrafish test results

  40. Sewage effluent - toxicity

  41. Sewage effluent – estrogenicity (males: >80%) 4 3 2 Plasma VTG concentration (µg/mℓ) 1 0 Control H1 H2 H3 H4 H5 Females Exposure Groups

  42. River and dam water

  43. Final drinking water

  44. Estrogen analyses on sewage effluent (ng/ℓ)

  45. Estrogen analyses on river and dam water (ng/ℓ)

  46. Estrogen analyses on final drinking water (ng/ℓ)

  47. Triazine and p-nonyl phenol concentrations (ug/ℓ) in river and dam water

  48. Triazine and p-nonyl phenol concentrations (ug/ℓ) in final drinking water

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