What are Endocrine disruptors?

1. Determination of some Endocrine Disruptors in a Sewage Treatment Oxidation Pond and a Receiving Stream by High Performance Liquid Chromatography and Inductively Coupled Plasma-Mass SpectrometryN. TortoDepartment of Chemistry, Rhodes University, Grahamstown 6140, South AfricaA.O. Ogunfowokan, E.K. Okoh, and A.A.AdenugaDepartment of Chemistry, Obafemi Awolowo University, Ile-Ife, 220005 Nigeria

2. What are Endocrine disruptors? • Endocrine disruptors (sometimes referred to as hormonally active agents) are exogenous substances that act like hormones in the endocrine system and disrupt the physiologic function of endogenous hormones • Substances that stop the production or block the transmission of hormones in the body and often interfere with development • Endocrine disrupting compounds encompass a variety of chemical classes, including pesticides, compounds used in the plastics industry and in consumer products, and other industrial by-products and pollutants • A large number of environmental pollutants including alkylphenolic compounds, polychlorinated biphenyls, and heavy metals including lead, mercury, cadmium and arsenic have been shown to disrupt endocrine functions in animals (Susan and John, 2001; Wu et al., 2003; Lee et al., 2003).

3. Any Adverse Health Effects? • Endocrine disrupting chemicals have been proposed as a potential cause of a numerous human health problems such as: • Birth defects; alterations in sexual and functional development (Thomas and Colborn 1992); • neurologic disorders, diabetes mellitus, immunologic disorder (Smoger et al,1993) • early puberty in young girls (Colon et al., 2000;Zacharids et al.., 1970), • Breast cancer (Wolff et al., 1993, Steingraber et al.,1997, contribution to subfertility (Newbold, 1995), • Reduced physical stamina (Guillette,et al., 1998) • Reduced sperm counts and un-descended testes (Toppari et al.,1996) and • Enlargement/reduction of prostate (vom Saal et al., 1997). • Inter-sex in fish e.g. fish from River Tames in London

4. Occurrence of Endocrine Disruptors (Phthalates) • Phthalates are components of many consumables, including: • Personal- care • Paints • Industrial plastics, and • Certain medical devices and pharmaceuticals (ATSDR, 1993, David et al., 1999). • Surface waters of south western Nigeria (Ogunfowokan and Fatoki 1993a&b; Ogunfowokan et al. 2006; Torto et al. 2007) • Phthalates are moderately persistent and, as a consequence of their wide use, are the most abundant man-made chemicals in the environment (Jobling et al., 1995). • They are not chemically bound to the polymer matrices of the products but are present as a mobile component, significant migration of them into the environment is inevitable.

5. Occurrence of Endocrine Disruptors (Lead, Cadmium and Arsenic) • Sources of Lead include: paint, Inks and dyes, Vehicular emission, Plastics and chemicals, Dust in the roof void etc (http://www.lead.org.au/fs/fst2.html) • Cadmium is released into the environment from mining and metal processing operations, burning fuels, making and using phosphate fertilizers, and disposing of metal products. • Arsenic is found in the natural environment in some abundance in the Earth’s crust and in small quantities in rock, soil, water and air. It is present in many different minerals. Industrial processes such as mining, smelting and coal-fired power plants are other sources

6. What are Phthalate Esters? • Phthalates are dialkyl or alkyl aryl esters of phthalic acid with the general structure shown below where R1 and R2 can be various combinations of straight and branched alkyl chain or aryl group. • Are persistent organic pollutants (POPs) and liable to undergo significant biomagnification • Chemicals that have high environmental toxicity to humans and other organisms

7. Health Effects of Pb, Cd and As Lead • For pregnant women, elevated Pb concentrations increase the risk of hypertension and birth defects (Rabinowitz, 1988); reduction in the IQ of Children (Ogunfowokan et al. 2000). Cadmium • Health effects of Cd are: Ittai-Ittai diseases, it also has mutagenic, carcinogenic and teratogenic effects (Fischer, 1987; Friberg, et al., 1986; Heinrich, 1988). Arsenic • Health effects of Arsenic are: decreased production of red and white blood cells, skin changes and lung irritation, infertility and miscarriages with women, and it can cause skin disturbances, heart disruptions and brain damage with both men and women, inorganic arsenic can damage DNA (http://www.lenntech.com/periodic-chart-elements/as-en.htm#ixzz0QXN5ykWO) • Humans may be exposed to Arsenic, Lead and Cadmium through food, water and air.

8. Generally, endocrine disruptors have the potential to mimic, or in some cases block, the effects of the endogenous hormone. They are therefore, described as endocrine disrupting chemicals, hormone disruptors or estrogen mimickers (Roberts, 1999; Colon et al., 2000). They are not under normal control and cause unregulated activities, hence in some cases there may be hyper-function (excessive function) or hypo-function (under function) The main target of the endocrine disruptors is the endocrine system

9. ENDOCRINE SYSTEM The endocrine system is instrumental in regulating metabolism, growth, development, puberty, tissue function and also plays a part in determining mood Endocrine system Endocrine system Major endocrine glands: 1.Pineal gland2.Pituitary gland3.Thyroid gland4.Thymus5.Adrenal gland6.Pancreas7.Ovary8.Testes. Source: http://en.wikipedia.org/wiki/Endocrine_system

10. Our Early efforts on Endocrine disruptors • Development of methods for the quantitative determination of Phthalate Esters in Surface waters, sewage treatment oxidation pond and Tap Water (Fatoki and Ogunfowokan 1993a &b, Ogunfowokan et al., 2006) • Other work from the study area includes the determination of some physicochemical parameters in the sewage treatment oxidation pond (Ogunfowokan et al. 2005 & 2008) Fatoki, O.S. and Ogunfowokan, A.O. (1993b): Environment International 19, 619-623 Fatoki, O.S. and Ogunfowokan, A.O. (1993a): International Journal of Environmental Studies: 44, 237-243. A.O. Ogunfowokan,, N.Torto , A.A.Adenuga and E.K.Okoh (2006): Environmental Monitoring and Assessment 118 , 457-480 The Netherland) N. Torto, Lesego C. Mmualefe, J.F. Mwatseteza, B. Nkoane, L. Chimuka, M.M. Nindi and A.O. Ogunfowokan (2007): Journal of Chromatography A, 1153: 1–13 Ogunfowokan A.O., Adenuga A.A., Torto N., E.K.Okoh (2008)Environmental Monitoring and Assessment. The Netherland 143: 25-41 Ogunfowokan A.O., E.K. Okoh, A.A. Adenuga and O.I. Asubiojo (2005):Journal of Applied Sciences 5 (1): 36-43.

11. Why this study? • Endocrine disruptors are of concern because of their subtle toxicity effects since they affect the normal function of the endocrine system • Small but critical changes in the chemical makeup of an environment are enough to trigger outcomes that could lead to population decline and loss of bio-diversity • This study therefore, focused on the identification and quantification of some phthalate esters and some heavy metals that have been implicated as endocrine disruptors in a sewage treatment oxidation pond; as a source and the impact on a receiving stream.

12. Study area and sampling points Figure 1 is the map of the study area showing the sampling sites. Measurement points from the sampling sites have been designated S1 to S8. Sample SR represents the reference point upstream before discharge of the effluent into the stream and serves as control. The Obafemi Awolowo University campus has two Oxidation ponds A and B lying side by side. (Figure1). Each pond measures 150m by 32m, and is about 1.2m deep. A 5m wide dyke separates the ponds. The wastes are conveyed to the ponds through a network of concrete pipes of different diameters. Only one pond receives influents and waste water at a time. The sewage is retained in the pond for about 2 weeks during which algae, bacteria and other microorganisms act on them. Each pond has a discharge channel through which effluents are discharged into the receiving stream. Site S1was located 30m away from the inlet to the pond, before the influent enters the pond. S2 was located at the inlet to the pond and S3 was located right on the pond about 50m away while site S4 represents the point of exit of effluents from the oxidation pond. S5 was a point located at the place where the pond's effluent enters the receiving river while sites S6 and S7 were located down stream, at approximately 15m and 100m respectively away from S5. S8 was located about 350m away from the confluence of Opa River and the receiving stream, which is about 800m away from S5. Site SR was the reference point and was located about 20m up stream from the point of discharge of the effluents into the receiving stream (S5).

13. S4 S1 S3 S5 B S2 S6 S7 Abattoir S8

14. Chemicals and Reagents All reagents used in this study were of analytical grade and were further purified before use. Ultra pure water was from a Milli-Q system supplied by Millipore (Bedford, MA, USA). N-hexane and ethyl acetate were from Ultrafine Limited, (Finchely,London). Acetonitrile was from Riedel-de Haën (Sigma-Aldrich, Laborchemikalien GmbH, Germany). Other reagents used were: sodium chloride from Associated Chemical Enterprises, (Glenvista, RSA), sodium carbonate from SAARCHEM, (Muldersdrift, RSA), anhydrous sodium sulphate and dichloromethane were obtained from Rochelle chemicals (Johannesburg, RSA) while aluminum oxide was from Fluka Chemica (Switzerland). HNO3 and HClO4 were obtained from BDH Chemicals (Poole, England). Sample Handling Samples for phthalate esters analysis : were collected in 2.5liter amber Winchester glass bottles, which had been thoroughly washed and rinsed with triply distilled water, oven dried and finally rinsed with triply distilled dichloromethane. The bottles were rinsed thoroughly with the samples before they were finally filled. The mouths of bottles were covered with glass stopper after filling them with samples to avoid contamination from plastic covers. Alteration of the organics due to microbial activities was prevented by acidification of the samples to pH 2 with concentrated hydrochloric acid immediately after collection and samples were stored in refrigerator at about 40C prior to analysis. Samples for metals analysis : were collected using plastic bottles that have been previously cleaned by washing in detergent, rinsed with tap water, and later soaked in 10% HNO3 for 72 hours and finally rinsed with deionised water prior to usage. During sampling, sample bottles were rinsed with sampled water several times and then filled to the brim. The samples were transported to the laboratory immediately and stored in the refrigerator at about 4ºC prior to analysis. Experimental

15. Extractions Ultra pure water was used as the blank sample. 1000mL aliquot of ultra pure water was measured into a 2 L beaker and acidified with concentrated HCl to pH 2, then saturated with about 20g of NaCl. This was extracted three times with 15ml of CH2Cl2 each time. The CH2Cl2 extracts were combined and they contained phthalate esters and other organic contaminants. The free fatty acids (FFA) interferences were removed by further extraction with 3 x 10mL 0.1M Na2CO3. The organic extracts after alkali washing, were then dried over anhydrous Na2SO4. The dichloromethane was subsequently evaporated by purging with nitrogen gas. The same procedure was employed for real samples. Sample clean-up A 10 mL column was packed with about 12.5g of activated alumina prepared in a slurry form in n-hexane. The residue extracts were redissolved in 2ml CH2Cl2 and chromatographed through the packed column. Hydrocarbons and phthalate esters were eluted successively from the column with 20mL of n-hexane and 30mL ethyl acetate. The ethyl acetate eluate was concentrated to 1ml by purging with nitrogen gas. This was diluted with 1mL acetonitrile for LC/MS analysis. Trace Organics analysis (Phthalates)

16. Quality assurance study Quality assurance was carried out by recovery experiments in order to ascertain the precision and efficiency of the analytical procedure. This was done by extracting a sample spiked with 10 mL of a mixture of five authentic phthalate esters at a concentration of 1 mg/mL. Using a spiked sample has the limitation that some analytes strongly retained on the particles may not be extracted. However, since no certified reference material was available to us when this study was conducted, this method was used for method validate.

17. Analyses of Metals Digestion of water samples The samples were digested using mixture of acids. The method adopted was described by Carrondo et al. (1979). 10mL of water sample was put in a pretreated Teflon beaker and 30ml of concentrated HN03 was added and the mixture was evaporated to dryness on a hot plate in a fume cupboard. This was allowed to cool and 5mL HNO3, 2mL 60% HClO4 and 6mL 40% HF acids were added. The resulting mixture was evaporated to dryness, and then 2mLHNO3 and HClO4 acids were added and evaporated to dryness to ensure that silicon and fluoride were removed. The final residue was re-dissolved in 2.5mL of 2M HNO3 and brought up to the mark of 25mL standard flask with distilled water. A blank digestion was as well carried out for background correction. The worked-up samples above were analyzed using ICP-MS.

18. Phthalate Ester Determination of phthalate esters was achieved after separation using a HP1100 series HPLC system, Agilent Technologies (Waldbronn, Germany), equipped with a diode array detector and thermostated column compartment. Chromatographic separation was carried out using a 250mm x 4.6mm i.d. Kromasil 100 C18 column with particle size of 10μm, from SUPELCO (Bellefonte, USA). Separation was performed under gradient elution conditions using acetonitrile and water as mobile phase, with an injection volume of 25 µl and flow rate of 1 ml/min, with the column temperature was set at 40 C. The elution gradient started with 50% acetonitrile, which was increased linearly to 75% over four minutes. Then over four minutes, it was changed to 100%, and the condition was maintained for twelve minutes before returning to initial percentage in four minutes Metals The metals As, Cd, and Pb were analyzed after acid digestion using a Finnigan MAT Element 2 High Resolution Inductively Coupled Plasma - Mass Spectrophotometer Finnigan (Bremen, Germany). For ICP-MS analysis, the isotopes of the elements determined were; 111Cd, 75As and 208Pb. The RF power was 1.158kW, nebulizer gas flow rate was 1.0L/min, cooling gas flow rate was 14.89L/min, with a detector voltage 2398V. Instrumental

19. RESULTS AND DISCUSSION

20. % Recovery of Phthalate Esters from Spiked Water Samples

21. Figure 2: Representative Chromatogram of mixture of Phthalate Esters obtained on HPLC

22. Levels of phthalate Esters in the Oxidation pond and the receiving Stream USEPA criteria of 3μg/L phthalates recommended for the protection of fish and other aquatic life in water and the SNAEL of 7.5–38.5μg/L for drinking water. This should give cause for great environmental concern. Peoples’ health downstream is at stake and so is the ‘health’ of the ecosystem.

23. Interpretation of infrared spectrum of phthalate esters in samples from Sewage treatment oxidation pond

24. Mass Chromatogram of Dibutyl Phthalate Ester

25. Mean values for metals in the sewage treatment oxidation pond

26. Average values for metals in the receiving stream

27. Average values for metals in the reference samples

28. oxidation pond receiving stream reference samples Maximum allowable threshold of metals in water intended for human consumption and that will not constitute a threat to health: As = 10 μg/L (DWAF 1996); Pb = 100 μg/L (FEPA); Cd = 3 μg/L (WHO 1993)

29. Conclusion • The levels of phthalate ester plasticizers and metals were studied in a sewage lagoon effluents and its receiving stream in this study. Liquid–liquid extraction and high performance liquid chromatography and Inductively Coupled Mass Spectrometry have been used for quantitative analysis of phthalate esters and metals. • The results obtained showed high levels of phthalate esters, As, Cd and Pb in the sewage lagoon and the receiving stream with the former being higher in phthalates and metals than the latter. • The discharge of the effluent from the sewage lagoon into the receiving stream led to increase in the concentrations of the analytes downstream and has therefore impacted the receiving stream.

30. Conclusion/Recommendation This is unfortunate because lives of fish and aquatic biota in the receiving stream are at risk and the rural dwellers that depend on the water from the receiving stream for various domestic purposes downstream untreated are at great risk of serious health effects due to phthalate esters and metals pollution. The sewage lagoon serves as point source pollution into the receiving stream. There is therefore a need for urgent action by the authority in charge of the sewage lagoon to upgrade it to improve its treatment performance. Thorough analysis and study of water from the stream before being used for domestic applications is therefore recommended to minimize the health risk. Future work should be carried out on fish and sediments of the receiving stream to ascertain biomagnifications of these analytes in these matrices.

31. Food for thought !!!! • Therefore, my beloved brethren, be ye steadfast, unmoveable, always abounding in the work of the LORD, for as much as ye know that your labour is not in vain in the Lord (I Corinthians 15 verse 58) KJV

32. Acknowledgement I would like to appreciate the Local organizing committee of the Humboldt International conference and Stiftung /Foundation for the full sponsorship granted to me

33. Thank You all for your attention !!!!!!!!