Appropriate Microbial Indicator Tests for Drinking Water in Developing Countries and Assessment of Ceramic Water Filters by Chian Siong Low B.A.Sc., Civil and Environmental Engineering University of Toronto, 2001 Submitted to the Department of Civil and Environmental Engineering in Partial Fulfillment of the Requirements for the Degree of Master of Engineering in Civil and Environmental Engineering at the Massachusetts Institute of Technology June 2002 ' 2002 Massachusetts Institute of Technology All rights reserved Signature of Author(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133) Department of Civil and Environmental Engineering May 13, 2002 Certified by(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133) Susan E. Murcott Lecturer, Department of CEE Thesis Supervisor Accepted by(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)............... Oral Buyukozturk Chairman, Department Committee on Graduate Studies Appropriate Microbial Indicator Tests for Drinking Water in Developing Countries and Assessment of Ceramic Water Filters by Chian Siong Low Submitted to the Department of Civil and Environmental Engineering on May 13, 2002 in Partial Fulfillment of the Requirements for the Degree of Master of Engineering in Civil and Environmental Engineering ABSTRACT Indicator organisms such as coliforms and E.coli frequently replaced pathogens in the monitoring of microbial quality of drinking water. Tests for indicator organisms are typically easy to perform and results can be obtained quickly. Many studies have concluded that total coliform is not an appropriate indicator in tropical environments. Instead, E.coli is a better indicator of recent fecal contamination and E.coli is proposed as the indicator organism of choice for routine water monitoring in developing countries. Two Presence/Absence (P/A) tests were studied and compared to Membrane Filtration (MF). The P/A-Total Coliform test is useful in evaluating disinfected water supplies. The P/A-H S- 2 producing bacteria test is simple, inexpensive, and suitable for monitoring microbial quality of drinking water in the rural areas. The MF test allows the enumeration of indicator organisms and can be used to assess the microbial removal efficiencies of point-of-use water filters. Different culture media for various indicator organisms were compared based on cost, ease of result interpretation, and medium preparation. The author concluded that m-ColiBlue24 be used for total coliform detection, m-FC with rosalic acid for fecal coliform detection, and either EC with MUG or m-ColiBlue24 for E.coli detection. For point-of-use water treatment, the author also fabricated a ceramic disk filter in collaboration with Hari Govinda Prajapati, a local pottery maker in Thimi, Nepal. The manufacturing process was documented and design improvements were recommended. Two of these ceramic filters were brought back to MIT and evaluated. Two other Indian TERAFIL terracotta ceramic filters were also tested in the laboratories in Nepal and MIT. Both TERAFIL filters consistently removed 85% turbidity and produced water with less than 1.0 NTU. Total coliform, fecal coliform, and E.coli removal rates exceeded 95% with one exception. However, the two TERAFIL filters have very different maximum flow rates of 2 and 7 L/hr. The Thimi ceramic filters have similar turbidity and microbial removal rates. However, they have significantly lower flow rates of 0.3 L/hr. Despite the high microbial removal rates, some form of household disinfection is necessary for these filters if zero coliform count is to be achieved. Thesis Supervisor: Susan E. Murcott Title: Lecturer, Department of CEE ACKNOWLEDGEMENTS I would like to express my most sincere thanks to: Susan Murcott, my thesis supervisor, for all her guidance and support throughout my entire Nepal project and thesis. I really appreciate her untiring proofreading of my long thesis and the invaluable feedback she has given me. She has been a true inspiration and mentor all this while. Everyone in ENPHO, to all the Misters, Misses, Didi, Dai, for all the help in the lab and field. My experience in Nepal will never be as complete and enjoyable without you all. Hari Govinda, for all your assistance and enthusiasm with the ceramics while I was in Thimi. Fellow M.Eng(cid:146)ers, for sticking it out with me for the whole semester! AY, RC, AW, VL, KL, FY, JS, ET, AC, KC in Toronto. Ur e reason y I(cid:146)m here, n still alive. I(cid:146)ll miss u all. Esp. to AY, RC, KL: Thx 4 all e listening. U noe how much they all mean to me. (SB: This 9 mths r for u too.) And most importantly, my parents, and my two brothers and their families. I would not have come so far if not for all the care, support, and the peace of mind you provided me all these years. 3 TABLE OF CONTENTS CHAPTER 1 : INTRODUCTION........................................................................................................11 1.1 Motivation for Microbial Indicator Study..........................................................................11 1.2 Filtration as Point-Of-Use Water Treatment......................................................................12 1.3 Field Studies in Kathmandu, Nepal....................................................................................13 1.4 Study Objectives.................................................................................................................16 CHAPTER 2 : WATERBORNE PATHOGENS AND DISEASES..........................................................17 2.1 Introduction to Waterborne Diseases..................................................................................17 2.2 Significance of Pathogens in Drinking Water Supplies......................................................18 2.3 Four Main Classes of Pathogens.........................................................................................19 2.4 Indicator Organisms of Drinking Water.............................................................................23 CHAPTER 3 : SUITABILITY OF COLIFORMS AS INDICATORS........................................................30 3.1 Introduction to the Coliform Indicator................................................................................30 3.2 Why Coliforms are Chosen as Indicators...........................................................................31 3.3 Why Coliforms are Unsuitable Indicators..........................................................................34 3.4 Inappropriate Use of Coliforms as Fecal Indicators in Tropical Environments.................38 3.5 Proposed Drinking Water Monitoring Methodologies in Tropical Developing Countries 39 CHAPTER 4 : PRESENCE/ABSENCE INDICATOR TEST.................................................................43 4.1 P/A Test for Coliform Indicator..........................................................................................43 4.2 P/A Test for Total Coliform and E.coli..............................................................................43 4.3 Water Sampling and Testing Methodology........................................................................44 4.4 Sampling Procedures for P/A-Total Coliform Test............................................................45 4.5 Identification of Total Coliforms with Varying Reactions.................................................47 4.6 Indicator Organisms Isolated from P/A-Total Coliform Test.............................................49 4.7 Sensitivity of P/A-Total Coliform Test..............................................................................50 4.8 Summary of P/A-Total Coliform Test................................................................................52 CHAPTER 5 : ANOTHER PRESENCE/ABSENCE INDICATOR TEST................................................53 5.1 P/A Test for H S-producing Bacteria.................................................................................53 2 5.2 Rationale for Developing the H S Test...............................................................................53 2 5.3 Preparation of H S Test Medium........................................................................................53 2 5.4 Sampling Procedures for H S Test.....................................................................................54 2 5.5 Association of H S-producing Bacteria with Coliforms and Fecal Contamination...........55 2 5.6 Indicator Organisms Isolated from H S Test......................................................................58 2 5.7 Sensitivity of H S Test........................................................................................................59 2 5.8 Effect of Incubation Temperature on H S Test..................................................................62 2 5.9 Summary of H S Test.........................................................................................................65 2 CHAPTER 6 : MEMBRANE FILTRATION INDICATOR TEST.............................................................66 4 6.1 Methods of Microbial Enumeration....................................................................................66 6.2 How Membrane Filtration Works.......................................................................................66 6.3 Advantages of Membrane Filtration over Multiple Tube Fermentation Method...............67 6.4 Methodology of MF Test....................................................................................................67 6.5 Sampling Volumes for TC/FC/E.coli Tests........................................................................70 6.6 Classical Metabolic Methods of Coliform Detection.........................................................72 6.7 Enzymatic Methods of Coliform Detection........................................................................74 6.8 Modified Membrane Filtration Culture Media for Total Coliform....................................76 6.9 Selecting Culture Media for Different Indicator Organisms..............................................78 6.10 Total Coliform Media (cid:150) m-Endo, m-ColiBlue24fi, Chromocultfi....................................79 6.11 Fecal Coliform Media (cid:150) m-FC with rosalic acid, EC.......................................................82 6.12 E.coli Media (cid:150) m-ColiBlue24fi, EC with MUG...............................................................84 6.13 Summary of Culture Media Recommendations for Membrane Filtration........................86 CHAPTER 7 : MANUFACTURING CERAMIC WATER FILTERS IN NEPAL.......................................88 7.1 Selection of Ceramic Filters in Nepal.................................................................................88 7.2 Local Ceramics Cooperative in Thimi................................................................................88 7.3 Making A Ceramic Filter in Thimi.....................................................................................90 7.4 Filter Manufacturing Procedure..........................................................................................93 CHAPTER 8 : ASSESSMENT OF CERAMIC WATER FILTERS........................................................99 8.1 Two Filters Studied: TERAFIL and Thimi Ceramic Filters...............................................99 8.2 Indian TERAFIL Terracotta Ceramic Filter.......................................................................99 8.3 Thimi Terracotta Ceramic Filter.......................................................................................101 8.4 Other Studies on the TERAFIL........................................................................................101 8.5 Methodology of Filter Testing..........................................................................................103 8.6 Variations in Test Conditions...........................................................................................105 8.7 Test Results and Discussion.............................................................................................109 8.8 Correlation of Results.......................................................................................................116 8.9 Filter Tests Summary........................................................................................................120 8.10 Recommendations for Future Work...............................................................................121 CHAPTER 9 : CONCLUSIONS AND RECOMMENDATIONS............................................................122 REFERENCES..............................................................................................................................125 APPENDIX A – ADDITIONAL TABLES AND DRINKING WATER GUIDELINES AND STANDARDS..134 APPENDIX B – MANUFACTURING PROCEDURES OF SOME OTHER CERAMIC FILTERS............138 5 LIST OF TABLES Table 1-1: Summary of indicators for Nepal................................................................................14 Table 2-1: Waterborne disease outbreaks in the United States, 1980 to 1996.............................17 Table 2-2: Causes of waterborne disease outbreaks in USA, 1981-1990.....................................19 Table 3-1: Identification of coliforms isolated from drinking water on LES ENDO agar...........33 Table 3-2: Non-community water systems: comparison of coliform monitoring results prior to and after an outbreak.............................................................................................................34 Table 3-3: Correlation coefficients for coliform bacteria, turbidity and protozoa in a watershed. ...............................................................................................................................................35 Table 3-4: Relationship between percentage of coliform antagonists and the presence of coliforms...............................................................................................................................37 Table 3-5: Relationships between different indicators as extracted from different literature sources...................................................................................................................................38 Table 3-6: Number of indicator bacteria commonly found in human feces (Wet Weight)..........39 Table 4-1: Frequency of reactions in P/A bottles and their confirmation rate for TC..................47 Table 4-2: Confirmation efficiencies of TC with P/A, MPN, MF techniques..............................48 Table 4-3: Effect of increasing coliform numbers on indicator bacteria combinations and on the response time to produce a presumptive positive P/A result................................................48 Table 4-4: Distribution of organisms isolated from raw, drinking, and water from new mains by P/A tests................................................................................................................................49 Table 5-1: Agreement of positive H S tests with various indicator tests (cid:150) A cross comparison 2 between studies.....................................................................................................................56 Table 5-2: H S-producing bacteria isolated from drinking water samples...................................58 2 Table 6-1: Suggested sample volumes for MF-TC test................................................................71 Table 6-2: Suggested sample volumes for MF-FC test................................................................71 Table 6-3: Performance summary of tests carried out with m-ColiBlue24fi medium on TC and E.coli recovery......................................................................................................................77 Table 6-4: Different coliform colony colors with different culture media...................................79 Table 6-5: Summary of TC culture media in terms of cost, ease of result interpretation, and medium preparation..............................................................................................................82 Table 6-6: Summary of FC culture media in terms of cost, ease of result interpretation, and medium preparation..............................................................................................................84 Table 6-7: Summary of E.coli culture media in terms of cost, ease of result interpretation, and medium preparation..............................................................................................................86 Table 6-8: Summary of selected MF culture medium to use for each indicator organism...........86 Table 7-1: Proportions of red clay, sawdust, and rice husk ash used in the first set of prototypes fired at 1000°C......................................................................................................................91 Table 7-2: Chemical composition of pottery clay used in Thimi.................................................93 Table 8-1: Summary of TERAFIL performance as tested by five different laboratories...........102 Table 8-2: TERAFIL filter test performance under lab conditions............................................110 Table 8-3: TERAFIL and Thimi ceramic filter test performance under lab conditions.............111 Table 8-4: Correlation coefficients of various performance parameters for TERAFIL (MIT)..116 Table 8-5: Correlation coefficients of various performance parameters for TERAFIL (ENPHO). .............................................................................................................................................116 Table 8-6: Performance summary of TERAFIL and Thimi ceramic filters...............................120 6 LIST OF FIGURES Figure 1-1: Map of Nepal.............................................................................................................14 Figure 2-1: Waterborne pathogen classifications.........................................................................21 Figure 2-2: Indicator organism classifications..............................................................................24 Figure 3-1: Relationship between disease risk and viruses, coliforms and FC............................32 Figure 3-2: Relationship between disease risk and Salmonella, coliforms and FC......................32 Figure 3-3: Study of the survival and multiplication of coliforms and faecal streptococci in relatively unpolluted lake waters..........................................................................................36 Figure 3-4: Regrowth of coliforms and E.coli in sewage effluent after inactivation with 5mg/L chlorine.................................................................................................................................36 Figure 3-5: Persistence of selected enteric bacteria in storm water stored at 20(cid:176)C......................36 Figure 4-1: HACH LT/BCP 20ml glass ampule...........................................................................43 Figure 4-2: General sampling and testing methodology of the author.........................................44 Figure 4-3: P/A equipment and supplies for TC test....................................................................45 Figure 4-4: Different reactions with the P/A broth when TC are absent or present in various concentrations after 48 hours................................................................................................46 Figure 4-5: Fluorescence of the P/A broth after 48 hours in the top most of the 3 bottles when E.coli is present in the water sample.....................................................................................46 Figure 4-6: Presence and Absence TC results compared to MF-TC test enumeration.................51 Figure 5-1: HACH PathoScreen P/A media pillow and box........................................................54 Figure 5-2: P/A test equipment and supplies for H S bacteria test. 100 ml sample bottle shown in 2 picture...................................................................................................................................54 Figure 5-3: Absence and presence results of the H S test after 24 or 48 hours............................55 2 Figure 5-4: Illustration of the relationships between TC, FC, and H S bacteria..........................58 2 Figure 5-5: Presence and absence H S results compared to MF-TC test enumeration................60 2 Figure 5-6: Presence and absence H S results compared to MF-FC test enumeration.................61 2 Figure 5-7: Presence and absence H S results compared to MF-E.coli enumeration...................61 2 Figure 5-8: Left sample was incubated at 35”C for 24 hours and some black color can be seen at the bottom. MF results show 9 TC per 100ml. Right sample showed a positive H S Test 2 with TC exceeding 600 CFU per 100ml...............................................................................63 Figure 5-9: Effects of temperature and FC concentration on incubation period..........................64 Figure 6-1: Millipore glass MF setup with Millipore incubator on the left..................................68 Figure 6-2: Portable Millipore stainless filter holder....................................................................68 Figure 6-3: m-Endo medium showing dark red coliform colonies with metallic sheen...............79 Figure 6-4: m-Endo medium showing a few coliform colonies with metallic sheen, but also with many background colonies which makes counting difficult.................................................79 Figure 6-5: Plastic ampules are pre-packed with 2 ml of (from left to right) m-Endo, m- ColiBlue24fi, m-FC media from Millipore...........................................................................79 Figure 6-6: m-ColiBlue24fi medium showing coliform colonies as red colonies and E.coli (only one E.coli colony) as blue colonies.......................................................................................80 Figure 6-7: m-ColiBlue24fi medium showing a sample crowded with blue colonies (E.coli) and red colonies (TC). Despite the overcrowding, the colonies still show up distinctly which makes counting possible. Brown background is a result of a high iron content in the water sample...................................................................................................................................80 7 Figure 6-8: Chromocultfi agar medium showing coliform as salmon pink colonies and E.coli as blue colonies (overcrowding)...............................................................................................81 Figure 6-9: m-FC with rosalic acid medium showing FC as distinctive blue colonies with little interference...........................................................................................................................82 Figure 6-10: EC medium showing FC as cream colored colonies that are less distinctive compared to the m-FC medium............................................................................................82 Figure 6-11: EC with MUG medium (looks exactly the same as the EC medium) prepared from BD/Difco powdered medium................................................................................................83 Figure 6-12: EC with MUG medium showing E.coli colonies fluorescing under a long- wavelength (366nm) ultraviolet lamp...................................................................................85 Figure 6-13: E.coli colonies on a EC with MUG medium not under a ultraviolet lamp..............85 Figure 7-1: Traditional (cid:147)Potters Wheel(cid:148) using an old tire and spinning it by hand with a stick..89 Figure 7-2: Pottery making in open courtyards where finished pots are left to dry.....................89 Figure 7-3: Pots ready to be fired in the traditional way are covered with hay and ash...............89 Figure 7-4: Pots are fired between 3-5 days in covered ash mound with small side vents emitting smoke....................................................................................................................................89 Figure 7-5: White clay candle filter..............................................................................................90 Figure 7-6: ENPHO arsenic ceramic filter....................................................................................90 Figure 7-7: Cutting the bottom part of the plastic containers purchased from marketplace.........92 Figure 7-8: Filter disk placed in the plastic containers and silicone applied all around for water sealing...................................................................................................................................92 Figure 7-9: Three basic raw materials (from left to right) (cid:150) Red pottery clay, rice husk ash, and sawdust..................................................................................................................................94 Figure 7-10: Hari measuring the various proportions using a green bowl...................................94 Figure 7-11: Proportions mixed in a red plastic basin..................................................................94 Figure 7-12: Mixture placed in a plaster mold made by Hari. The mold has an inner diameter of 6(cid:148) and depth of 3(cid:148).................................................................................................................95 Figure 7-13: Excess is scrapped off to form a smooth surface after pressing and filling the mixture to the top..................................................................................................................95 Figure 7-14: The mold is carefully inverted to remove the mixture and is labeled for easy identification.........................................................................................................................95 Figure 7-15: Mixtures allowed to dry for 5-7 days before firing..................................................96 Figure 7-16: Dried mixtures are placed in the kiln and fired at a temperature of 1000-1070°C for 12 hours.................................................................................................................................97 Figure 7-17: Filters after firing and ready to be affixed. Lighter color in filters after firing........97 Figure 7-18: 6-inch diameter ceramic containers also fabricated by Hari....................................97 Figure 8-1: TERAFIL filter tested in MIT..................................................................................100 Figure 8-2: TERAFIL filter tested in ENPHO............................................................................100 Figure 8-3: TERAFIL ceramic filter disk...................................................................................100 Figure 8-4: Two Thimi ceramic filters with ceramic filter disks of different compositions that are brought back to MIT...........................................................................................................101 Figure 8-5: Top view of the upper container showing the ceramic filter disk A........................101 Figure 8-6: Simplified diagram showing the top container of the TERAFIL filter and water level. .............................................................................................................................................104 Figure 8-7: Simple diagram showing the top container of the Thimi ceramic filter and water level.....................................................................................................................................104 8 Figure 8-8: TERAFIL (MIT) showing the original white cement used to bond the ceramic filter to the metal container..........................................................................................................106 Figure 8-9: TERAFIL (MIT) showing the silicone added on top of the white cement after drying. .............................................................................................................................................106 Figure 8-10: Location near Harvard bridge where water samples are collected from the Charles River....................................................................................................................................108 Figure 8-11: Collecting river samples from a (cid:147)very(cid:148) polluted Dhobi Khola River in Kathmandu, Nepal...................................................................................................................................108 Figure 8-12: Comparison of the Dhobi Khola River sample with distilled water......................109 Figure 8-13: Collecting high turbidity water from a well near the ENPHO lab.........................109 Figure 8-14: Two graphs plotting the flow rates vs. turbidity removal rates of TERAFIL (MIT) and TERAFIL (ENPHO)....................................................................................................119 9 LIST OF ABBREVIATIONS µm Micrometer BCP Bromocresol Purple BGLB Brilliant Green Lactose Bile cm Centimeter CFU Colony Forming Unit Chromocult Agar for simultaneous detection of Total Coliform and E.coli CRW Charles River Water DST Defined Substrate Technology E.coli Escherichia Coli EC Escherichia Coli ENPHO Environment and Public Health Organization FC Fecal Coliform g Gram H S Hydrogen Sulfide 2 HPC Heterotrophic Plate Count IBDG Indoxyl-β-D-glucuronide ID Infective Dose LT Lauryl Tryptose MAC Maximum Acceptable Concentration MF Membrane Filtration MF-E.coli E.coli Membrane Filtration Test MF-FC Fecal Coliform Membrane Filtration Test MF-TC Total Coliform Membrane Filtration Test MI Agar for simultaneous detection of Total Coliform and E.coli MPN Most Probable Number MTF Multiple Tube Fermentation MUG 4-methyl-umbelliferyl-β-D-glucuronide m-ColiBlue24 Medium for simultaneous detection of Total Coliform and E.coli m-Endo Medium for detection of Total Coliform m-FC Medium for detection of Fecal Coliform m-TEC Medium for detection of E.coli mg Milligram ml Milliliter NGO Non-government Organization NRs Nepali Rupee (US$1 = NRs 75) ONPG o-nitrophenyl-β-D-galactopyranoside P/A Presence/Absence P/A-H S Hydrogen Sulfide Producing Bacteria Presence/Absence Test 2 P/A-TC Total Coliform Presence/Absence Test POU Point-Of-Use Rs Indian Rupee (US$1 = Rs 45) TC Total Coliform TSA Tryptic Soy Agar TTC Triphenyltetrazoliumchloride USEPA United States Environmental Protection Agency WHO World Health Organization X-Glu/BCIG 5-bromo-4-chloro-3-indolyl-β-D-glucuronide 10