1. Pathogen removal and effluent reuse Universidade Federal de Viçosa Departamento de Engenharia Civil Rafael K.X. Bastos 8 th IWA SPECIALIST GROUP CONFERENCE ON WASTE STABILIZATION PONDS 2 nd Latin-American Conference on Waste Stabilization Ponds Belo Horizonte, Brazil, 26-30 April 2009 current state of the art knowledge gaps knowledge gaps future research future research

2. Pathogens is wastewater OrganismConcentration Escherichia coli10 6 -10 8 /100 mL Salmonellae spp.10 2 -10 3 /100 mL Giardia cysts10 2 -10 4 / L Cryptosporidium oocysts10 1 -10 2 / L Helminth eggs10 1 -10 3 / L Viruses10 2 -10 5 / L

3. Rotavirus Protozoa (oo)cysts (4 – 20 μm) Viruses (nm) Helminth eggs ( > 50 μm ) Bacteria ( ≈ 1 μm ) Pathogens characteristics Salmonella Giardia Cryptosporidium Ascaris

4. Pathogens removal in WSP Long HRT Inactivation Sedimentation http://www.personal.leeds.ac.uk/~cen6ddm/

5. WSP design for pathogens (bacteria) removal Dispersed flow Complete-mix (cells in series ) Complete-mix (one cell) Plug flow EquationSchematicHydraulic Regime Source : von Sperling (2007)

6. Marais (1974) First order kinetics complete-mix reactors K FC : temperature dependent die-off rate: the same in anaerobic, facultative and maturation ponds Von Sperling (1999), Polprasert & Bhattarai (1985), Agunwamba et al. (1992) First order kinetics Dispersed flow rectors Dispersion numbers (d) Advances on WSP design for bacteria (faecal coliforms) removal

7. Curtis & Mara (1994) Environmental factors Mara (2002) Anaerobic ponds von Sperling (1999) Pond depth Advances on WSP design for bacteria (faecal coliforms) removal

8. Influent faecal coliform Temperature Design flow Hydraulic retention time Die-off rate constant (…) uncertainty of the input design parameters vs deterministic average single values random values selected from a range (uniform probability distribution) Monte Carlo Simulation  random value design procedure repeated for any required number of times.  design outputs: frequency histogram and cumulative frequency curves (decision-making) (e.g.: 95%ile of FC < 1,000 / 100 mL) Advances on WSP design for bacteria (faecal coliforms) removal

10. Sensitivity analysis which input design parameters significantly influence the design output von Sperling (1996, 2002) one input design parameter is allowed to vary within a proposed range the rest: single average values Advances on WSP design for bacteria (faecal coliforms) removal

11. Gawasiri (2003)

12. Advances on WSP design for bacteria (faecal coliforms) removal

13. K b (dispersed flow) = - 0.7118 Ln (H) + 0.8589 (20 o C)R 2 = 0.4491 K b (dispersed flow) = 0.542 H - 1.259 (1)Von Sperling K b (dispersed flow) = 0.917 (H) - 0.877. (HRT) - 0.329 (2)Von Sperling Bacteria (faecal coliforms) removal in WSP knowledge gaps & future research (local contexts) Observed and estimated values of effluent E.coli concentrations using von Sperling models, equation 1 (left) and equation 2 (right). Bastos et al (2009)

14. Water (half-depth) x air temperature WSP design for bacteria (faecal coliforms) removal knowledge gaps & future research (local contexts) Brito (1997) Rios (2008) Belo Horizonte, Brazil (UFMG) Viçosa, Brazil (UFV)

15. Bastos et al (2006) Organism Kb (20 o C) (d -1 ) Pond 1Pond 2Pond 3 Samonella sp.4.572.603.00 E.coli2.731.412.20 Pathogens (bacteria) removal in WSP

16.  Well designed and properly operated and maintained WSP systems can achieve a 3-6 log unit removal of bacterial pathogens, and a 3-4 log unit removal of viruses (WHO, 2006)  10 3 FC / 100 mL effluent quality ► absence of pathogenic bacteria Salmonellae, Campylobacter, Vibrio cholerae, Shigellae… (!!??) Pathogens (bacteria and viruses ) removal in WSP knowledge gaps & future research  Enteroviruses, Norovirus, Rotavirus, Adenovirus, Astrovirus, Hepatitis A virus, Hepatitis E virus, Polyomavirus… (???) Viruses (????) Oragui et al. (1987, 1993)

17. WSP design for parasites removal Ayres et al (1992) http://www.personal.leeds.ac.uk/~cen6ddm/

18. Bastos et al (2006) Pathogens (parasites ) removal in WSP knowledge gaps & future research (local contexts)

19.  Well designed and properly operated and maintained WSP systems can achieve a 1-2 log unit removal of protozoan (oo)cysts, and a 3-4 log unit removal of helminth eggs (WHO, 2006)  < 1 (human) nematode egg / L effluent quality ► absence of other settlable organisms [eggs and (oo)cysts] (??) Protozoan (oo)cysts (Giardia and Cryptosporidium) (!!??) Grimason et al (1993) Microsporidia (Enterocytozoon bieneusi and Encephalitozoon Intestinalis), Cyclospora cayetanensis, Toxoplasma (…) (??) Pathogens (parasites ) removal in WSP knowledge gaps & future research

20. Pathogens (parasites ) removal in WSP knowledge gaps & future research Ascaris lumbricoides Ascaris suun Toxocara canis GiardiaCryptosporidium Bevilacqua et al (2008)

21. WW reuse http://www.personal.leeds.ac.uk/~cen6ddm/ Lins - SP Approaches to setting microbiological guidelines (i)The absence of faecal indicator organisms in the wastewater (ii)No measurable excess cases in the exposed population (iii)A model-generated risk which is below a defined acceptable risk Blumenthal (2000)

22. Type of reuseTreatmentEffluent quality Unrestricted irrigation ‘zero risk’ (?) Secondary + filtration + disinfection BOD  10 mg/L Turbidity  2 NTU Chorine residual  1mg/L Faecal coliforms ND Pathogens ND Restricted irrigation risk (?) Secondary + disinfection BOD  30 mg/L Chorine residual  1mg/L Faecal coliforms  200/100 mL Effluent quality for WW reuse USEPA (2004)

23. Type of reuseNematode eggs/LFC / 100 mL Unrestricted irrigation (*) < 1< 10 3 Unrestricted irrigation (**) < 1- WHO (1989) Effluent quality for WW reuse ‘Best available’ epidemiological evidence * workers’ risk ** workers’ and consumers’ risk Ponds !!! Ponds !!! Helminths Bacteria, protozoa Viruses Risk ranking (theoretical model)

24. Effluent quality for WW reuse (WHO, 2006) Options for the reduction of viral, bacterial, and protozoan pathogens by different combination of health protection measures that achieve the health-based target of 10 -6 DALYS pppy (risk-based approach!!)

25. Effluent quality for WW reuse (WHO, 2006) Pathogens reduced by treatment and post-treatment (pre-ingestion) health-protection control measures (i) Method of wastewater application; (ii) die-off between last irrigation and consumption; (iii) food preparation (washing/peeling)

26. Effluent quality for WW reuse (WHO, 2006) QMRA Epidemiological evidence

27. Effluent quality for WW reuse (WHO, 2006) viral, bacterial, and protozoan infections: guidelines based on tolerable additional disease burden of ≤10 -6 DALYS (disability-adjusted life year) loss per person per year (pppy) Quantitative microbial risk analysis (QMRA)

28. QMRA : Dose – response + exposure scenarios Hass et al (1999) Microrisk (2006)

29. Farmer wades through homemade diversion canal, which carries wastewater to his fields in Pakistan. Source: IWMI (2003). Harvesting watercress from a wastewater canal in Vietnam. Source: IWMI (2003). labour intensive x highly mechanized agriculture labour intensive x highly mechanized agriculture QMRA : Dose – response + exposure scenarios

30. QMRA - Effluent quality for WW reuse (WHO, 2006) exposure scenarios

31. Advances on QMRA - Effluent quality for WW reuse http://www.personal.leeds.ac.uk/~cen6ddm/

33. QMRA - Effluent quality for WW reuse knowledge gaps & future research (exposure scenarios)

34. QMRA - Effluent quality for WW reuse knowledge gaps & future research (exposure scenarios)

35. QMRA - Effluent quality for WW reuse knowledge gaps & future research (exposure scenarios)  Constant ratio of pathogen numbers to E. coli numbers (??)  0.1–1 rotavirus and Campylobacter per 10 5 E. coli  0.01–0.1 Cryptosporidium oocyst, per 10 5 E. coli  Pathogen die-off (reduction) between harvest and consumption (??)  10 -2 -10 -3 rotavirus and Campylobacter  0–0.1 oocyst

36. QMRA - Effluent quality for WW reuse knowledge gaps & future research (tolerable risk) http://www.personal.leeds.ac.uk/~cen6ddm/ Tolerable risk (?) ◄► WW effluent quality (treatment level) Tolerable risk (?) ◄► WW effluent quality (treatment level)

37. Pathogens removal in WSP - QMRA - Effluent quality for WW reuse knowledge gaps and future research (detection methods) Detection methods for enteric pathogens  Detection methods for enteric pathogens  Culture dependent methods  Bacteria: viable but nonculturable bacteria (VBNC)  Cell culture monitoring for cytopathogenic effect (CPE), enzyme linked immuno assay (ELISA) ► some viruses, such as norovirus, cannot be cultured in vitro.

38. Detection methods for enteric pathogens  Detection methods for enteric pathogens  Microscopy  Helminths and protozoa: concentration, immunomagnetic separation, fluorescence microscopy ► laborious, expensive and inaccurate.  Fluorescent in situ hybridization (FISH) (bacteria, protozoa) ► require expensive equipment and highly trained staff. S. Enteritidis Oliveira e Bernardo (2002) Pathogens removal in WSP - QMRA - Effluent quality for WW reuse knowledge gaps and future research (detection methods)

39. Detection methods for enteric pathogens  Detection methods for enteric pathogens  Nucleic acid based methods  amplification (Polymerase Chain Reaction) (PCR) of target DNA or reverse transcription followed by PCR (RT-PCR) for target RNA, quantitative real time PCR (qPCR), denaturing gradient gel electrophoresis (DGGE)(viruses, protozoa, bacteria) ► require expensive equipment, standardization and highly trained staff.  amplification (Polymerase Chain Reaction) (PCR) of target DNA or reverse transcription followed by PCR (RT-PCR) for target RNA, quantitative real time PCR (qPCR), denaturing gradient gel electrophoresis (DGGE) (viruses, protozoa, bacteria) ► require expensive equipment, standardization and highly trained staff. Enteroviruses in pond effluent (Venezuela) Guastadisegni et al (2002) Pathogens removal in WSP - QMRA - Effluent quality for WW reuse knowledge gaps and future research (detection methods)

40. Pathogens removal in WSP - QMRA - Effluent quality for WW reuse knowledge gaps and future research Remarkable advances !! More field data Thank you !!! On-going research  Pathogens removal (mechanisms) in ponds ► robust (simple) design models  Pathogens reduction (treatment + post-treatment) ► QMRA ◄► epidemiological evidence