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Aquatest: An Affordable Water Test by Robert Bain (G) Fourth-year undergraduate project in Group D, 2007/2008

“I hereby declare that, except where specifically indicated, the work submitted herein is my own original work”

Signed ………………………………………..

Date:…………………

Technical Abstract
Aquatest: An Affordable Water Test

Motivation and purpose The project was motivated by the scale and severity of problems with water supplies in developing countries. Every year 1.8 million deaths (of these 90% are children under five) are attributable to poor water supplies, sanitation and hygiene (WHO, 2004). Despite considerable effort by the international community to tackle this problem, success has been limited; a different approach is clearly needed. Community-driven development is easier to sustain, but the technology to facilitate the shift of power has not been developed. The monitoring of water quality is a case in point; laboratories are centralised and portable testing kits based on Membrane Filtration are too expensive (around £1000) for most communities to afford. The purpose of this project is two-fold: (i) To examine water supply, sanitation and hygiene in rural areas of developing countries and determine whether bacteriological water testing is appropriate for this context. And if there is a need; (ii) To develop an affordable and easy to use bacteriological water testing device that enables widespread water quality monitoring in rural areas. Is water testing appropriate? An initial literature review reinforced the need for water testing: “Surveillance and verification of drinking water quality in small community water supplies is recognised as an essential activity; however it remains potentially complex and expensive”
International network on small community water supply management Alice Springs, Australia July 2005

However, further exploration cast doubts on the appropriateness of water testing. A combination of case studies, interviews and a thorough literature review were used to establish the need for water testing, assess its limitations and gain a better understanding of the context. Aquatest: An Affordable Water Test | Technical Abstract i

Whilst water testing can be inappropriate, it has been found that there is a pressing need for an affordable test. This need is clearest for young children and HIV/AIDS suffers for whom exposure to faecal contamination represents a significant health risk. Aquatest and the design of a low-cost device In January 2008 contact was established with Aquatest, an international consortium developing a low-cost water test. Tasks assigned to this project were: determining requirements for this device, evaluating incubation methods and developing design concepts. Working with Aquatest greatly increased the potential of this project. In addition to the above tasks, the literature surrounding statistical inference of bacteriological assays (the “most probable number” or MPN) was reviewed and a model to determine the operational characteristics of the device was developed. Once the exposureresponse relationship is known, this model can be used to validate design concepts. Main Findings (i) Water testing can be inappropriate in some cases even if a functional and affordable device were developed; water quality can be a low priority for adults who have strong immunities. (ii) There is little evidence to justify the use of quantitative water testing as more than an indicator of the extent of contamination. The causal link needs to be firmly established before it can be considered a measure of the health risk. (iii) Water testing is strongly linked to Household water storage and treatment (HWST) and the importance of source-to-point of use contamination. Care must be taken not to further the water quality agenda over the other preventative measures such as improving access to water, hygiene education and sanitation facilities. (iv) An affordable water testing kit using the MPN sample subdivision, rather than membrane filtration method can be developed. A simple design concept has been proposed. This uses body heat to incubate the water sample (rather than £600 electronic incubators) and a disposable pipette to transfer water into the device. Aquatest: An Affordable Water Test | Technical Abstract ii

Table of Contents
Technical Abstract ................................................................................................................. i List of Figures ................................................................................................................. iiv List of Tables .................................................................................................................. iiv List of Boxes ..................................................................................................................... v Acronyms ......................................................................................................................... v 1 Introduction ...................................................................................................................... 1 2 Summary of the Project .................................................................................................... 3 3 Methodology ..................................................................................................................... 6 4 Part I: Exploratory Research ........................................................................................... 10 4.1 Case Studies ............................................................................................................. 10 4.2 Significance of Water Quality .................................................................................. 10 4.3 Alternative Measures............................................................................................... 14 4.4 Can Testing Lead to Action?..................................................................................... 16 4.5 Evaluation of Current Water Testing Practices........................................................ 17 4.6 Conclusions .............................................................................................................. 22 5 Part II: Constructive Research ......................................................................................... 24 5.1 Determining Requirements ..................................................................................... 24 5.2 Critical Evaluation of the Aquatest Prototype ......................................................... 27 5.3 Experimental methods ............................................................................................. 28 5.4 Concepts and Their Evaluation ................................................................................ 39 6 Final Conclusions............................................................................................................. 41 6.1 Findings .................................................................................................................... 41 6.2 Recommendations and Further Work ..................................................................... 42 6.3 Critical Evaluation of the Project ............................................................................. 42 7 Appendix ......................................................................................................................... 43

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List of Figures
Figure 1: Areas of Risk for Traveller’s Diarrhoea ................................................................. 1 Figure 2: Rotavirus distribution ........................................................................................... 1 Figure 3: Methodology Flow Diagram ................................................................................. 7 Figure 4: Faecal-Oral transmission paths........................................................................... 11 Figure 5: Categories of monitoring and interventions ...................................................... 14 Figure 6: Hydrogen Sulphide test ...................................................................................... 19 Figure 7: IDEXX Quantitray 2000 ....................................................................................... 20 Figure 8: Schematic of the relationship between indicator groups .................................. 21 Figure 9: Requirements Capture ........................................................................................ 24 Figure 10: Functional Diagram ........................................................................................... 24 Figure 11: Functional analysis of incubation ..................................................................... 25 Figure 12: Incubation temperature and time to get a result with the H2S test ................ 25 Figure 13: Model of Aquatest first device prototype ........................................................ 27 Figure 14: Likelihood plots for a single dilution with fifty wells. ....................................... 31 Figure 15: Discrimination between threshold values using Monte Carlo ......................... 33 Figure 16: Heating and heat storage options that were considered ................................. 35 Figure 17: Incubation using body heat .............................................................................. 35 Figure 18: Bessel Function ................................................................................................. 38 Figure 19: Experiments to determine critical radius ......................................................... 38 Figure 20: Equivalence between empty submerged tube and suspended filled tube ...... 38 Figure 21: Final concept waterjet demonstration model .................................................. 40 Figure 22: Final design concept CAD drawing ................................................................... 40 Figure 23: Pipettes or sterile syringe ................................................................................. 40 Figure 24: Graph of Coefficient of Variation...................................................................... 45 Figure 25: Plots demonstrating the Most Probable Range ............................................... 46 Figure 26: Random number allocation .............................................................................. 46

List of Tables
Table 1: Comparison of deductive and inductive research ................................................. 6 Table 2: Criteria for water testing to be considered appropriate ....................................... 9 Table 3: Summary of Case Studies ..................................................................................... 10 Table 4: Description of water testing methods ................................................................. 17 Table 5: Evaluation of testing kits against criteria ............................................................. 20 Table 6: Problem Definition ............................................................................................... 24 Table 7: Requirements Specification ................................................................................. 26 Table 8: Advantages of MPNS over MF ............................................................................. 27 Table 9: Statistics Nomenclature ....................................................................................... 29 Table 10: Statistical assumptions....................................................................................... 29 Table 11: Discrimination between example threshold values for a 50ml sample ............ 33 Table 12: Morphological Chart .......................................................................................... 39 Table 13: Case Study Findings............................................................................................ 44 Table 14: Measures of Uncertainty ................................................................................... 45

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List of Boxes
Box 1: The Aquatest Initiative Box 2: Occupancy Theory

Acronyms
HIV/AIDS IWA JMP MIT MPN MPN MPNMD MPNS MTF UNICEF WHO Human Immunodeficiency Virus/ Acquired Immune Deficiency Syndrome International Water Association Joint Monitoring Program Massachusetts Institute of Technology Most Probable Number Most Probable Number MPN-Multiple Dilution MPN-Sample Subdivision Multiple Tube Fermentation United Nations Children's Fund World Health Organisation

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1 Introduction
1.1 Motivation
The motivation for the project is the scale and severity of disease related to problems of poor water quality in developing countries. According to estimates 1.1 billion people in developing countries do not have access to an “improved” supply of water; of these 84% live in rural areas (JMP, 2006). Faecally contaminated water is one of the main transmission routes of infectious diseases and poses a major threat to human health. Every year 1.8 million people are killed by diarrhoeal disease resulting from a lack of clean water, inadequate sanitation and poor hygiene. Ninety percent of these are children under the age of five (WHO, 2004). These figures are likely to underestimate the true extent of the problem as a significant proportion of the disease goes unrecorded (IWA, 2003); they also overlook the morbidity caused by diarrhoeal disease which is aggravated by restricted access to healthcare, malnutrition and the prevalence of HIV/AIDS. Diarrhoeal disease primarily affects those with undeveloped or weakened immune systems: children, the elderly and HIV/AIDS suffers (and travellers). Water-borne diseases are often perceived as an entirely developing country problem (fig. 1). They are also affected by climate and are particularly prevalent in tropical countries; the warmer, wetter and more humid climates tending to exacerbate problems with water quality and sanitation (fig. 2).

Figure 1: Areas of Risk for Traveller’s Diarrhoea (Centre for Disease Control and Prevention, 2008)

Figure 2: Rotavirus distribution of cases. Each point represents 500 out of a total of 800,000 yearly deaths (Glass, 1997)

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1.2 Approach
A new approach to monitoring is needed Despite considerable effort and investment over the years, the international agenda to improve water and sanitation has not seen the success expected. Furthermore, it seems unlikely that the Millennium Development Goal for water (#7 Target 10: “halving the number of people without sustainable access to safe drinking water”) will be met in many countries – the number of people without access in Sub-Saharan Africa is in fact increasing (JMP, 2006). As noted by the World Health Organisation the top down approach has limitations: ‘While global estimates of coverage will remain important, local capacity to generate and use information will be a vital part of the monitoring effort’ (WHO, 2008) The most sustainable approach would be to reduce the dependence of the poor on external support through community-based or driven development. In many cases the infrastructure and technology to facilitate this shift of power has not been made available; one area which is particularly lacking is the monitoring of water supplies. This project assesses one way of monitoring water quality: bacteriological water testing referred to as water testing below. Water testing Bacteriological water contamination comes from many sources – much of which has little sanitary significance. The purpose of water testing is to detect recent faecal contamination (Hutton, 1983) and identify water that is unfit for drinking. It is not practical to routinely test for all pathogenic micro-organisms that might be in drinking water; instead tests should detect bacteria that are indicative of faecal pollution. Faecal-oral disease transmission is responsible for the spread of water-borne disease and therefore the presence of these indicator micro-organisms is thought to give a good assessment of the health risk posed by drinking water. If indicator bacteria are not present then the water is deemed safe to drink. The three indicators that are often used are coliforms and more specifically thermotolerant coliforms or Escherichia coli (known as E.coli). Bacterial densities are usually given as a number per 100ml – the volume of water usually tested. Aquatest: An Affordable Water Test | 1 Introduction 2

1.4 Project Objectives
(i) To examine water supply, sanitation and hygiene in rural areas of developing countries and determine whether water testing is appropriate in this context. And if there is a need; (ii) To develop a low-cost and easy to use bacteriological water testing device that enables widespread water quality monitoring in rural areas of developing countries.

2 Summary of the Project
Familiarity with a low-cost water test developed at Massachusetts Institute of Technology (MIT) led the author to the initial conclusion that an appropriate water test could be produced for a fraction of the current price. Experience using this kit at a school in Tocantins, Central Brazil reinforced the perceived need for monitoring water quality. The boarding school (Fundação Bradesco – Canuanã) wanted to continue testing water as an educational exercise in nearby communities, but was unable to because the test relies on several consumables that are expensive and not available in rural areas (Section 6.3).

“Surveillance and verification of drinking water quality in small community water supplies is recognised as an essential activity; however it remains potentially complex and expensive”
International network on small community water supply management Alice Springs, Australia July 2005

A brief literature review at the start of the project supported the need for low-cost water testing (see quote above), however as the project progressed the benefits of water testing were increasingly called into question. The initial proposal for the project was to further develop the work carried out on the MIT water testing kit and to make it suitable for community use. It quickly became clear that whilst this would result in a relatively low-cost kit (at around 1/10 of the cost of commercially available testing kits such as DelAgua) it would remain out of the reach of people earning less than $2 a day. A previous 4th year project (Gordon, 2006) had followed this path, achieving only incremental improvements to the low-cost kit.

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Part I: Is water testing appropriate for rural water supplies? In light of the limitations of such a project, contact was made with organisations for which an appropriate test could be developed or where the issues of water quality could be researched. The first of these was the Karen Hilltribes Trust, a charity which builds gravityfed water supplies in Northern Thailand. The charity was contacted with the intention of designing a low-cost water test that was appropriate for the Karen (one of the ethnic hill tribes). Telephone conversations and interviews with the head of the charity led to several interesting findings and the overall conclusion was that water testing was not appropriate for the Karen. There were very good reasons for this including: • • Mountain springs have little to no contamination and with adequate treatment (Biosand filtration) the water was known to be clean. Volunteers had attempted to conduct water testing for the charity, but it had been plagued by procedural difficulties and inconclusive results. Attempts to get in contact with organisations in Northern Mozambique where water quality problems are particularly acute (and because the author speaks Portuguese) were also unsuccessful. It became clear that research would have to be conducted in the UK and would have to rely primarily on secondary sources. It was decided to build on these unexpected findings, explore case studies from secondary sources and to conduct a thorough literature review in order to establish when water testing is appropriate. The initial research questions were: 1 2 3
Where does water testing fit into strategies for the (improvement of) provision of clean drinking water? Is testing appropriate? If so when, and what can it achieve? What are the most important factors to measure? What are the best ways to do these?

The case study research highlighted further concerns (technical and socio-political) and even called into question the basic assumptions on which water testing is based (Section 4.2.1). Interviews and secondary sources supported these findings. Whilst water testing is seen as inappropriate in several circumstances, it has been found that there is still a pressing need for a low-cost device (Section 4.6.2).

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Part II: Aquatest In January 2008 contact was established with the Head of the Aquatest Initiative, Dr. Stephen Gundry (Box 1). Working for Aquatest marked a significant change in the direction of the project and greatly increased both its scope and potential. Large-scale manufacture together with the development of a new field testing kit opened up the possibility of producing a device at a cost that will be affordable to those in need. The research areas agreed with Dr. Gundry were: Determining requirements for the device Evaluating methods for incubating the device Developing design concepts

With the results of the research outlined in Part I and criticisms of water testing in mind, work began on these tasks (Section 5). And in addition, a statistical model was developed to ascertain the number of wells and volumes needed to discriminate between a given set of risk levels (Section 5.3.1).

Box 1: The Aquatest Initiative Aquatest is a collaborative effort to design and disseminate a low-cost water testing kit for developing countries. It is led by the University of Bristol and collaborators include WHO, UC Berkeley and the University of Cape Town and Indian Participants. With support from the Gates Foundation ($13.1 Million), they aim to develop a small singleuse device for an ambitious cost of $0.10. The test is to be used by water professionals and communities themselves. It is hoped that ‘within 10 years, low-cost water testing devices will be widely used in 80% of developing countries (Aquatest website)’. The first prototypes will be field tested in India and South Africa in 2009. Distribution and Licensing of the technology will be led by PATH, an organisation that has experience with the disposable syringe.

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3 Methodology
‘most people become experienced with one type of study and then stick to it, safely and unadventurously’ (Hakim, 1992)

This project combines both deductive and inductive research: the former primarily in the first half, the latter predominantly in the second. The two approaches are compared in Table 1. The main weakness of the deductive approach, in which engineers are traditionally trained, is that it ‘eliminates the possibility of unanticipated findings’ (Eishenhardt, 1989).
Table 1: Comparison of deductive and inductive research

Deductive Starts from General Theory Form specific hypotheses Numerical or statistical Narrow Testing and confirmation

Inductive Starts from Observations Identify Patterns Textual Open-ended Explanation of reasons

Familiarity with the MIT water testing kit meant that approaching the problem with a new perspective was of utmost importance. Inductive approaches helped to avoid coming up with theories at the outset. In working for the Aquatest Initiative it is particularly important to avoid advocacy; only through questioning the benefits of water testing could substantive research be produced (Hakim, 1992). The methodology used is shown schematically in figure 3 on the next page.

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Figure 3: Methodology Flow Diagram

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3.1 Part I: Exploratory Research
Research was conducted predominantly through the use of inductive approaches: case studies, interviews and a thorough literature review. Case studies were central to the first stages of the project. Theory-building case study research is appropriate “in the early stages of research on a topic or to provide freshness in perspective to an already researched topic” (Eishenhardt, 1989). It can be seen that both of these applied to this research project: a fresh perspective on water testing was needed and little research had been done to establish whether tests are appropriate for use in rural areas of developing countries. Analysis was carried out in two parts: (i) individual caseanalysis and (ii) searching for cross-case patterns. Case studies guided the literature review and highlighted many issues with water testing. Interviews were informal. They ranged from telephone interviews with the head of the Karen Hilltribes Trust (KHT) to discussions with experienced Water and Sanitation and Public Health engineers. Several meetings were arranged with the Head of the Aquatest initiative. The Literature Review had several purposes: (i) to gain an understanding of the wider issues surrounding water quality in developing countries, (ii) to validate the case-study research findings and (iii) to pursue lines of enquiry. An appreciation of the wider context of water testing was seen as vital to ensure that the monitoring process is adequate, appropriate and most of all informative.

3.2 Part II: Constructive Research
The constructive research was heavily influenced by two sources: • • Good Design Practice for Medical Devices and Equipment (Shefelbine, 2002) The Inclusive Design Toolkit (Clarkson, 2007).

Conventional engineering design process focuses on conceptual design: “where the most important decisions are made” (Cross, 1994). The central preoccupation is seen to be not missing good ideas. In contrast, the primary focus of the process followed in this study was to establish the real need and ensure that the design solves a genuine problem.

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3.3 Criteria for Success: Defining “Appropriate”
It is interesting to note that whilst the Aquatest device will support grassroots development it goes against much of current development thinking. It will be mass manufactured and the technology is not easily understood by the user (they are not able to take control of the technology). The central notion of “intermediate technology” (from which the term appropriate technology was popularised) is that it addresses the need for labour intensive rather than capital intensive work; hence lending itself to small-scale, decentralised workplaces (Schumacher, 1973). The definition of “appropriate” has changed in the light of some very successful projects that have used modern mass-manufactured goods: examples include mobile telephones in Bangladesh and LEDs for rural lighting. In this report, appropriate simply means that it is suited to the specific needs of the users and the demands of the context (in this case rural areas of developing countries). The following criteria (Table 2) were developed during the project and have been used to determine whether water testing is appropriate in this context.

Table 2: Criteria for water testing to be considered appropriate

1. Relevant 2. Significant 3. Worthwhile 4. Affordable 5. Independent 6. Environmentally Sustainable 7. Scalable

Meaningful and sensitive to the user’s education, aptitude and organising skill. Relevant to their Of sanitary significance – established link to health outcomes it must be possible to use information from the test and this information should not be readily obtained from another source; It must be low-cost to run and have a small upfront cost It should not be reliant on external support, or materials that are not available locally. It must not generate excessive waste at the point of use. It must be scalable to facilitate widespread monitoring of drinking water

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4 Part I: Exploratory Research
Is Water Testing Appropriate for Rural Water Supplies?

4.1 Case Studies
Table 3: Summary of Case Studies

Organisation Karen Hilltribes Trust Escola Bradesco Student Project Vigyam Ashram

Project Gravity-fed water supply Solar water disinfection and water testing using the low-cost MIT testing kit Proposed water quality assessment Water testing using the multiple tube fermentation method.

Location Northwestern Thailand Central Brazil Tolla Islands, Indonesia Pabal, India

Abbreviation CS1 CS2 CS3 CS4

Table 3 summarises the case studies used at the beginning of the project. The first two are the authors own observations and experience and have been described briefly in the summary. The last two were gathered from secondary sources and personal communication. Conclusions from the case study research are given in the Appendix 7.2.

4.2 Significance of Water Quality
The extent of diseases related to water, sanitation and hygiene was described in the introduction. A question that needs to be asked is: “how much of the disease is attributable to water quality?” This has major implications for how diarrhoeal disease is tackled globally, but at the community level and for this project it needs to be asked in order to justify water quality testing which is inherently linked to interventions improving water quality.

4.2.1 The Importance of Water-borne Disease
The faecal-oral transmission route (fig. 4) is complex and there are many ways for pathogens to be passed from one person to the next. An important question for anyone trying to reduce diarrhoeal disease through environmental interventions is: “which are the dominant transmission paths?”

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Figure 4: Faecal-Oral transmission paths (Prüss, 2002)

Before examining the evidence, faecal-oral diseases need to be classified as water-borne or water-washed (Feachem, 1978): Water-borne diseases are those that are transmitted when contaminated water is drunk. They can be prevented by improving water quality. Water-washed diseases are caused by water scarcity; an inadequate supply of water inhibits personal hygiene: washing hands and food. Water-washed diseases can be prevented by increasing quantity of water without improving the quality. If all of the diarrhoeal disease was transmitted via the water-washed route, as was suggested by Prof. Cairncross in an interview, water quality interventions (and water testing) would not be worthwhile. A causative link between water quality interventions and diarrhoeal disease needs to be established in order to justify water testing. For this we turn to Epidemiology, the study of the factors affecting health in a population.

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Review of the epidemiological evidence for water quality Whilst there are many studies of water quality interventions, most have methodological flaws and decisions cannot be based upon them. For example in the most recent metaanalysis Clasen et al. (2007) rejected 947 out of a total of 979 studies reviewed. These methodological problems have been well documented (Blum D, 1983). Until recently, there was consensus that water quality and access were both important means of reducing diarrhoeal disease; access was seen as more important than bacteriological quality (Jensen, 2004). Interest in Household Water Treatment and Storage (HWTS) has led to several new studies showing strong support for water quality interventions in the household. In their review Fewtrell et al. (2005) found that water quality interventions at the household gave a mean reduction of 39% in morbidity, whilst those at the source only achieved 11%. Furthermore Clasen et al. (2007) found that water quality interventions need not be combined with hygiene and sanitation to be effective. Despite these encouraging findings, Prof. Cairncross is convinced that the water-washed route is dominant – water scarcity, not quality is responsible for the transmission of diarrhoea. Of the four blinded studies reviewed in the paper he co-authored with Clasen (2007) none showed reductions in diarrhoeal disease. Intervention studies which are not blinded are liable to bias – both because the researchers hope to show their treatment is effective and because communities are likely to understate their disease because they are grateful. There are two main camps: 1. Those that advocate water quality interventions in the household; 2. Those that advocate greater access to water; In conclusion, the epidemiological evidence for water quality interventions is easily challenged; there is no consensus as to the relative importance of these transmission routes. The relative predominance is likely to be dependent on many factors, e.g. in drier areas scarcity is likely to be dominant; in wetter and more populous areas waterborne disease may be significant.

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4.2.2 “Safe” Water
There are two common principles in definitions of safe water: (i) that the water is drinkable and (ii) that it poses little risk to health. A further principle needs to be added for a safe water supply: access to adequate quantities. As was seen in the previous section, there is considerable debate on the relative importance of quality and quantity. The guideline value for bacteriological quality of drinking water is 0 Coliforms (or E.coli) per 100ml (WHO, 2006). While only a guideline value, governments often set this as a standard to be met by all supplies despite not being affordable. This target is not achievable in most rural areas of developing countries and does not encourage the incremental improvements in quality and access that are needed to reduce diarrhoeal disease. Exposure-response Given the lack of consensus surrounding transmission paths, it is no surprise that there is little data on exposure-response curves. Drinking water quality testing in the west has been focused on ensuring the efficacy of treatment processes rather than measuring a health risk thus not demanding research on the quantitative relationship between indicator density and health outcomes. The lack of evidence is apparent when compared to the vast literature on the health risks associated with bathing waters. It also explains the WHO’s unwillingness to suggest suitable targets for rural areas of developing countries despite much criticism. In conclusion, there is a real need for more research before quantitative testing is meaningful for people drinking water from sources which are not disinfected. This is also needed for appropriate water quality standards to be set to readdress the bias towards water quality interventions described in the next section.

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4.3 Alternative Measures

Figure 5: Categories of monitoring and interventions

4.3.1 Monitoring
Health Outcomes are the most desirable indicators because they are directly what we aim to improve through interventions. Monitoring of the source and intervening processes is indirect and therefore only useful if causal links between the measure taken and the desired outcomes can be established. The main problem with measures by outcome is that they do not usually identify actions that the community can take or locate sources of pollution. Cholera epidemics, when all water is suspect and should be boiled are an obvious exception. As was found in section 4.2 even with a rigorous epidemiological study, it can be hard to isolate the impacts. If it was always possible to know by examining the source whether water is clean, then water testing would not be worthwhile. Unfortunately, in many cases you cannot tell whether water from a particular source is safe to drink, let alone whether it is clean at the point-of-use. This being said, there is much to be gained from perceptions of the quality of Aquatest: An Affordable Water Test | 4 Part I: Exploratory Research 14

water sources, the type of source and treatment administered and the state of the water supply. It is not surprising that the open wells tested in Brazil (CS2) were heavily contaminated whereas the mountain springs in Northern Thailand (CS1) were clean. Sanitary surveys are a formalized means of assessing the environmental risks – they usually constitute a series of questions with “yes” or “no” answers. A score, the number of “yes” answers gives an indication of the risk. Like perceptions of quality, the current forms do not necessarily correlate well with the actual contamination of the source. Region specific surveys can be developed once the importance of different risk factors is understood. Sanitary surveys are very important but require experience (Hofkes, 1983) and the user must be literate. One of the main advantages of a low-cost water test is that it can be used with little or no training and could therefore be scaled up rapidly. The main weakness of water testing is that it does not indicate possible sources of contamination or measures to improve water supplies. Combining water testing and sanitary surveys would overcome these difficulties, but it is not clear how this can be done without depending on expertise.

4.3.2 Interventions
Figure 5 shows that there are many interventions which can reduce the likelihood or severity of diarrhoeal disease. Preventative measures are more ethically acceptable than relying on treatment, especially when access to healthcare is limited (stores of Oral Rehydration Sachets are only currently reaching 38% of 0-5 year olds in the developing world (UNICEF, 2008)). Behavioural change in particular is often the most cost-effective way of reducing risk, but requires trained staff and is dependent on external support. It was generally accepted that too much emphasis has been put on water supplies (Thompson, 2001). Feachem (1978) suggests that donors and governments both have vested interests in water supply interventions and that there has been a degree of ‘wishful thinking’ about the benefits. With the recent findings, HWTS are becoming increasingly popular and considerable efforts are being made in their development and dissemination. Their development has been spurred on by very positive results in recent intervention studies and the finding that water from “improved” sources is readily contaminated before Aquatest: An Affordable Water Test | 4 Part I: Exploratory Research 15

use. There are many HWTS, these include: chlorination, solar water disinfection and ceramic filters. Water testing can play a role in increasing awareness of disease transmission, hygiene and encouraging greater care for personal environment. This was its purpose in Brazil (CS2). However, it is primarily linked to interventions to improve water quality – in particular HWTS.

4.4 Can Testing Lead to Action?
Whether water tests will lead to action to improve water quality depends on many factors. The technology to treat water is available and in many cases affordable: it is a matter of priorities, preferences and whether the test can be understood. Priorities: ‘Many people may not perceive diarrhoea as being more than an inconvenience unless it is severe and life-threatening’ (Howard, 2002). Water quality is often a high priority for women, especially mothers, but it is a much lower priority for men. Immunity means that it is easy for adults to perceive the risk of drinking water as low. If water quality is a low priority, it is very unlikely that a water test will be bought no matter how low-cost it is. Interpretation: As was found in several of the case studies, many people do not share the scientific concept of disease. Waterborne diseases are particularly prone to supernatural beliefs. Unlike natural causes, you can drink ‘bad’ water for a while before getting ill. There is also the difficulty of blaming water when there are so many other transmission routes. If the technology is not easily understood by the user, there is a high risk of misinterpretation. These will need to be addressed by field testing and through careful design. Preferences: Chlorination is heralded as a simple solution, however many people do not like the change in taste. As noted by Zoeteman (1980) the physical characteristics of water are often the most important for the user. More contaminated sources are drunk because of a better taste or because they are more convenient.

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4.5 Evaluation of Current Water Testing Practices
4.5.1 Methods
The traditional method for determining faecal-contamination is multiple tube fermentation. It will be referred to as most probable number by dilution (MPND) to distinguish it from the method used in the Aquatest Device which is MPN by subdivision (MPNS). MPND is a laboratory based method requiring substantial quantities of reagent, glassware, distilled water, an autoclave and large incubator (Tab. 4). Laboratory testing is particularly

problematic for remote areas of developing countries - transportation is unreliable and time consuming making it difficult to analyse samples within the recommended 6hrs (WHO, 2006). It is also much more expensive, requires high calibre staff and the distance (physically and organizationally) between central laboratories and rural villages means that it takes a long time before remedial action can take place (Hutton, 1983). The development of the Membrane Filtration (MF) and more recently chromagenic reagents have made field testing kits feasible. The incubation requirement is greatly reduced; for MF a single Petri dish needs incubation whereas many test tubes need incubation in the case of MPND. MF is used by almost all commercial field testing kits, with the exception of Presence/absence (P/A) tests which are a simplified form of MPND compromising a single undiluted tube.
Table 4: Description of water testing methods

Water is passed through a membrane (typically 45μm pores) leaving the bacteria on the filter. The filter paper is placed in a Petri dish and broth that selects bacteria of sanitary significance is added. The Petri dish is then incubated (24hrs) to allow Membrane these bacteria to form visible colonies. These colonies are then counted to find the Filtration number of indicator bacteria (Colony forming units- CFUs) that were present in the (MF) water sample. If there are many CFUs it may become difficult to count; water that is expected to be highly contaminated can be diluted prior to filtration in order to lower the number of CFUs to within the counting range (around 100 CFU). Most Probable Number by Dilution Measured volumes of the sample water are diluted and pipetted into a series of sterile tubes containing broth. The dilution series is tailored to the expected concentration of bacteria in the sample. Test tubes are incubated for (24-48hrs depending on the broth used) after which the bacterial density can be inferred

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(MPND)

from the tubes which show growth. The most likely number of organisms is called the “most probable number” and Standard Tables are available for most dilution series.

Presence/

Presence Absence is effectively MPND reduced to a single undiluted tube. The

Absence (P/A) presence absence test does not provide a quantitative determination. Most This is similar to the MPND method, but avoids dilution. Instead the sample is Probable Number by Subdivision tables are not always available because the configurations differ from MT-MPN. (MPNS) mixed with the reagent and then split into small wells. The bacterial density of the sample is inferred from the number of wells showing positive growth. Standard

4.5.2 Equipment
There is a wide variety of water testing kits on the market; however most are intended for users in developed countries. Examples of commercially available field testing equipment specifically designed for use in developing countries include the DelAgua water testing kit, H2S test and in development the MIT water testing kit. IDEXX Quantitray® is also described as this was the inspiration behind the Aquatest approach. DelAgua (MF) Developed at the University of Surrey in collaboration with Oxfam, DelAgua is probably the best known portable water testing kit. The kit was designed primarily for international aid workers and in particular humanitarian emergencies and reflects their needs. In addition to measuring bacteriological contamination, the testing kit can measure a variety of chemical properties of the water including residual Chlorine. The kit costs a total of £1327 (Institute, 2008) and approximately £0.30 per test. Even with significant subsidies it can rarely be afforded by local NGOs, let alone communities or households. The kit is cumbersome (weighing 10kg) and, requires training (3 days), several, often imported, consumables (methanol, filters, pads and reagents) and an electronic incubator - all barriers to its widespread use. The DelAgua kit is only suitable for users conducting regular testing.

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Hydrogen Sulphide Test (P/A) At a cost of around Rs. 10 (about 12p) (IWP, 2007) this test is the most affordable water test available. It was designed not to require incubation and has many advantages over the MF based testing kits. The major limitation with the H2S method is that it only indicates whether bacteria are present or not – it does not give a quantitative estimate. This is useful for ensuring that chlorinated supplies are working effectively because you would expect no indicator bacteria to be present. However, most rural

Figure 6: H2S test (courtesy of Susan Murcott)

water supplies are not disinfected and have some level of faecal contamination: P/A tests condemn too many supplies (IWA, 2003). Another problem with the H2S test is that it detects Hydrogen Sulphide producing spores, some of which do not indicate faecal pollution. For this reason it is not recommended by WHO (2002) – a more recent study indicates that H2S only correlates with E.coli for concentrations of over 1000/100ml (Gupta, 2007). MIT Low-cost water testing kit (MF) The MIT low-cost water testing kit is based on MF. The filtration device is compromised of a baby bottle and syringe. Sterilisation is avoided by using disposable sterile inserts. The need for reliable electric supply for incubation is circumvented by a phase change incubator: a primary alcohol is used as a heat store and releases heat at a constant temperature as it solidifies. Experience in Brazil suggests that the sterile inserts and reagents are not readily available. Furthermore, the reagent used in Brazil (Millipore mColiblue24) is expensive and requires refrigeration – clearly an alternative is needed if the testing kit is to be independent of electricity.

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IDEXX Quantitray® (MPNS) A serial dilution is equivalent to splitting a sample of water into small wells of varying sizes. This is the basis of the Quantitray® procedure (named MPNS in this report). This is a relatively new development in water testing practices and is more accurate and easier to use than MF. However, Quantitray® is by no means appropriate for rural areas. It needs an electronic incubator and heat sealer.

Figure 7: IDEXX Quantitray 2000 (www.idexx.com)

4.5.3 Evaluation of the Testing Kits

Table 5: Evaluation of testing kits against criteria

Criteria
1 2 Relevant Significant 3 4 Affordable 5 6 7 Independent Environmentally sustainable Scalable Worthwhile

DelAgua no training yes yes no no yes no

MIT no training yes yes no no uses many consumables no

H2S yes not recommended by WHO condemns too many supplies yes yes if reused or recycled Yes

MPNS Yes if E.coli or thermotolerant if counts are within the range. can be manufactured at low-cost Yes if reused or recycled Yes

The review of current testing equipment suggests that it will be possible to use the principle behind Quantitray (MPNS) to develop a water test at price close to that of the H2S test and is able to discriminate between contaminated sources.

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4.5.4 Choice of Indicator Bacteria and Reagents
Water contains many bacteria, most of which are harmless. The ability to isolate particular groups of bacteria that are indicative of a health risk is of particular importance to the development of a water test that meets the needs of the user. In the west, it is acceptable to use the Coliform (Total Coliforms) group because testing is used to measure the efficacy of treatment not to gauge health risk. An important distinction has to be made between an indicator and an index. This has implications for the amount of information that can be derived from microbiological water tests. Indicators are bacteria whose presence indicates faecal contamination. An index is an indicator whose concentration is directly related to the health risk (Gleeson, 1997). While many alternatives have been suggested, Escherichia coli (E.coli) is recognised as the best indicator of faecal contamination (WHO, 2006). It is by far the most common bacteria in the human gut (up to 109 per gram of faeces (Gleeson, 1997)) and is found in warm-blooded animal faeces.
Figure 8: Schematic of the relationship between indicator groups

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4.6 Conclusions
4.6.1 Discussion of the Limitations of Water Testing
It is much harder to justify quantitative water testing than was expected at the start of the project. The main criticism of water testing is the limited evidence supporting the link between indicator densities and health risk. To justify quantitative assessments of water quality, the link needs to be firmly established. Information from the water test may be used in hygiene education, but its primary use is to ensure the bacteriological safety of water in the household. It is therefore strongly linked to HWST and avoiding source-to-point of use contamination. Care must be taken not to further the water quality agenda over the other preventative measures that need support: access, hygiene education and sanitation. Whether water testing will be a priority for individuals and communities and the value they will put on testing is dependent on many factors – these will have to be determined by field studies. Experience in Brazil (CS2) suggests that there will be resistance where the test is not readily understood by the community or if the scientific concept of disease is not shared. There is a high risk of misinterpretation if the user is expected to understand the result, but not the means by which it is found.

4.6.2 Establishing a Need for Water Testing
There is no way for households and communities in rural areas of developing countries to be sure that the water they drink is safe. Whilst there is much to be gained from indigenous knowledge, perceptions of quality can be misleading; they could be exposing themselves to an unnecessary risk of illness. Water testing is attractive as one way of addressing the question: “Is this water safe to drink?” Combined with sanitary surveys, water testing would provide an effective means of monitoring rural water supplies where contamination is expected. This would allow users to both detect faecal contamination and identify its source. It would enable communities and individuals to take control over their own water supply; not to rely solely on foreign aid or

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the stretched government agencies which have clearly failed, in many cases, to provide water that is suitable for consumption. Several bacteriological water tests are commercially available, but none is suitable for community level testing. Current tests are both too complex and expensive or they condemn too many supplies to be of use in this context. Indeed, little has changed since Mara (1973) noted that: “In the majority of existing water supplies in developing countries (particularly in rural areas) facilities for the bacteriological examination of the finished water are absent.” There is a real need for widespread monitoring of water supplies in rural areas. Water testing could offer a means in which to do this in an affordable way and with less demand for training than other monitoring methods. There is a clear need for people who are looking after children, the elderly or immune-compromised to assess water quality. Those most at risk need to be able to identify ways to lower their exposure to the faecal pollution. In the opinion of the author, this need justifies the work in the second half of the project: working with Aquatest to develop a low cost water testing kit.

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5 Part II: Constructive Research
Development of an affordable water test

5.1 Determining Requirements
Following Shefelbine (2002) there are four processes that need to be carried out before the requirements specification can be completed (fig. 9).In the second half of this report, the focus of the design is on what is seen as the primary user: mothers. Uses by professionals and the trade-offs for greater coverage are also discussed.
Table 6: Problem Definition

WHO?

Individuals and water professionals (Village user, Local water engineer, community leaders and health workers, household).

WHAT? WHY?

Water quality test that is low cost, portable, reliable To test bacteriological quality of water

WHERE? Rural areas of developing countries: at
the source, in the home or in a clinic

WHEN?

When water quality unknown and for regular monitoring of supplies
Figure 9: Requirements Capture (Shefelbine, 2002)

Figure 10: Functional Diagram

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5.1.1 An example of functional analysis: Incubation
Figure 11: Functional analysis of incubation

The Aquatest initiative is in the process of developing a reagent for E. coli which gives a visual signal without the need for UV light. Since the reagent has not been developed yet the influence of temperature on incubation time is not known. Growth models were reviewed, but it became clear that there were too many variables. An alternative approach is simply to use known results from the H2S test (fig. 12) as a basis for setting the requirements. As many of the H2S bacteria are E.coli, this is seen as a good approximation.
120 100

Time (hours)

80 60 40 20 0 14 22 28 37 44

Temperature (°C)
Figure 12: Incubation temperature and time to get a result with the H2S test (J. Pillai, 1997)

Requirement: Temperature shall not drop below 22°C nor rise above 44°C during the 36 hours of incubation. The temperature should be close to 37°C.

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5.1.2 Requirements Specification
The full requirements specification is listed in Table 7. It is important to note that whenever possible, the requirements have been relaxed so that the device is not needlessly constrained.
Table 7: Requirements Specification
Sample

Flexible Sampling Representative Aseptic Low Skill Maintenance Ready Signal Interpretable Temperature Precision Reliability Sensitivity Significant Comparable Waterproof Transportable Storable Independent Robust

Sustainability Portable Adaptable Affordable Scalable Safe Materials Aesthetics

able to sample from all water supplies (accessible - at point of collection) able to test intrinsic water quality encourages representative sampling sample is collected without being contaminated by the user does not require more than one day of training. no training required does not require maintenance by user User given signal when when the test result is ready User able to tell whether the water is suitable for drinking by (i) a normal healthy adult (ii) children under 5, the elderly and immune-compromised/HIV. maintains a temperature of between 22 and 44°C for a duration of at least 36 hours incubation close to the optimal growth temperature 37 users warned if the incubation has failed able to discriminate between thresholds (to be set based on epidemiological data) gives the correct threshold 95% of the time when used correctly sensitive to 10/100ml sensitive to 5/100ml test for E.coli. Shall test for E.coli or Thermotolerant Coliforms (WHO) help to prioritise the contamination problem not leak contents or be affected by dampness or humidity. able to get the device to the regions where it will be used in a cost effective manner small enough to fit in a pocket no special storage requirements shelflife of at least three months; if sold in boxes (3 + 2n months). not reliant on sterilisation, electricity, distilled water or any other resource that is not available able to be dropped 2m onto rock without breaking difficult for someone to open up unintentionally or intentionally by a child resistant to shocks and vibrations during transport reusable not generate excessive waste easily carried to and from the source (upto 1km by foot). can be used in fixed laboratory incubators less than $0.30 at full scale manufacture $0.10 delivered to the customer, the Aquatest target price. costing <$50 for a prototype, <$2 at 1000, <$0.3 at 100,000 able to scale up to 1,000,000 able to adapt to increasing demand for the product not toxic or dangerous if released, or adversely affect the environment during manufacture, distribution, operation or disposal. compatible with reagent and not impede growth of E.coli appear reliable and instill confidence in user positive associations with healthcare and wellbeing

Functional

Use and Interpret

D W W D D W W D D D W D D D D W D W D D W D W D D W D W D D W D W W D W D D D W

Performance

Test Characteristics Incubate

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5.2 Critical Evaluation of the Aquatest Prototype P
“It's a small, single-use use device for testing water quality. It can be used in the field, without electricity or skilled technicians. It is being designed for use in developing countries at a target manufactured cost of 10 US cents.” cents (Aquatest Website)

Figure 13: Model of Aquatest first device prototype (Davis & Gundry, 2007)

1. The design is complicated and it is unlikely to meet the cost target. target An exothermic reaction (red) surrounded by an a insulating layer (yellow in fig. 14) is clearly not an efficient way of incubating the sample. 2. It is not certain whether this configuration is adequate. 3. The choice of MPNS is appropriate to the requirements and represents the step change needed to make water wa testing affordable. It has many advantages over MF, the most important of which are:
Table 8: Advantages of MPNS over MF

It t is easy to contaminate the samples samples when using MF and it requires training – ‘a skilled laboratory worker should receive a week’s training and have an opportunity for further

Lower Skill

individual practice before he undertakes membrane filter analyses’ (Mcjunkin 1976, in Hutton 1983). . This is somewhat of an exaggeration, but MF is not easy to to use: by comparison the Roben’s Institute offer a 3 day training courses for their DelAgua kit.

Affordability MPSS is more affordable than MF Reliability
The assay is more reliable because suspended growth inherently allows the resuscitation and growth of injured bacteria (Fujioka, 1997). It is possible to tailor the device to the concentrations that are expected in rural water

Range

supplies – this means it can detect higher concentrations than MF (max 200CFUs) without dilution.

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5.3 Experimental methods
5.3.1 Statistics for the Device
Progress was hindered without results from the Aquatest Initiative describing the configuration of wells that were needed in the device. In order to develop the concepts and preliminary designs an appreciation of the how the design would affect the operational characteristics of the test was required. Two first observations can be drawn about MPNS: 1. More information can be gained about the concentration for a given volume if the number of wells is increased. 2. If there is at least one bacterium in the sample the device will show growth in at least one of the wells. The sensitivity of the test is therefore related to the total volume of water in the sample. 5.3.1.1 Relevance of the Statistics and Ease of Interpretation The test must be easily interpreted and the result relevant to the user. An MPN table: a chart which tabulates the most probable number (an estimate of the bacterial density) for each configuration of positive wells is clearly not appropriate. The user is not interested in a count rate, but rather an indication of the level of risk from which a decision will need to be made. Often, this will be a drink/don’t drink decision. Both Quantitray® 2000 and the Aquatest prototype have used wells of different sizes. This may be confusing as it is not intuitive that the largest wells are the least important. It is suggested that the wells ought to be the same size – this will make interpretation easier and increase the information gained by the test. 5.3.1.2 Literature Review There is an extensive literature surrounding the most probable number. At first efforts were focused on determining the most probable or most likely number, but have since turned to the more difficult matter of defining a measure of the uncertainty of bacterial counts using the method. It is a Standard Method and has been used widely in microbiology and medicine since 191 (McBride, 2005). However, approximate MPN calculations, rounding Aquatest: An Affordable Water Test | 5 Part II: Constructive Research 28

conventions and different methods of calculating the confidence limits lead to considerable variability in the Standard Tables (McBride G. , 2003). The literature describes serial dilutions as used in MPNMD; this is equivalent to the method used in this project as long as the organisms are assumed to be randomly distributed. It is helpful to introduce some notation and review the assumptions on which the calculation of the MPN is based before describing the methods used:
Table 9: Statistics Nomenclature

R – Number of wells r – Number of positive wells s – Number of sterile wells n – Number of E. coli in sample V – Total volume of sample v – Volume of a single alquilot L (D|H) – Likelihood of the data given the hypothesis
Table 10: Statistical assumptions

Assumption Detects one or more organisms. Randomly distributed

Description
If there is at least one organism there is an obvious change which can be clearly and consistently identified. Bacteria are separate, not

Comments
There are many ways in which a test may fail to detect a viable organism. If the medium is poor it can require several bacteria and therefore underestimate contamination. Supposed to thoroughly mix the sample. It is possible to take a sub-sample that contains no bacteria, especially if concentrations are low. Good agreement was found between the exact

clustered and they do not repel each other The volume examined is a

method and the simpler Poisson methods at small relatively small portion of a large

Poisson theory

n. This is not the case when there are many wells sample which had been or if a series of different sized wells are used and thoroughly mixed such that the making this assumption tends to overestimate the volume of each alquilot is small MPN (McBride, 2005).

or

compared to the sample volume (v/V << 1). These assumptions have been rejected because as noted by Tillet (1995) ‘Samples… are often 300 ml

Binomially distributed
Similar to the Poisson assumption, but results in less

or less… a large proportion of this is put into the dilution series’. The assumption is even less valid if the whole sample is tested as is the case with the skew. (Beliaeff, 1993) Aquatest Device.

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5.3.1.3 Methods
Calculating the MPN Using Occupancy Theory Estimates for the Most Probable Number (Thomas 1942, Cochran 1950) are not suitable for large R or multiple dilutions (McBride, 2005). While Standard Tables, simple estimates and even an MPN Calculator (Blogett, 2003) are available – the exact MPN had to be calculated. Only with the likelihood function could measures of uncertainty be determined. The likelihood functions were evaluated directly from Occupancy Theory (box 2) using Matlab. It is relatively computationally intensive and the code had to be sped up in order to calculate the likelihood functions within a reasonable time. This was achieved by creating lookup tables thus reducing the number of times slow functions (e.g. binomial[], ln[]) were called. Most Probable Number lookup tables were created to in order to evaluate the operational characteristics described in section 5.3.1.6. The lookup tables were compared to Standard tables to verify the technique. Box 2: Occupancy Theory (David, 1962) If n bacteria are randomly distributed among R test tubes of equal volume, the likelihood that (R-r) tubes will not receive any of these bacteria and therefore remain sterile is: ௥ ܴ ‫ݎ‬ ‫ݎ‬−݅ ௡ ‫ܮ‬ሺ‫ܴ|ݎ‬, ݊ሻ = ൬ ൰ ෍ ቈቀ ቁ ሺ−1ሻ௜ ൬ ൰ ቉ ‫ݎ‬ ݅ ܴ
௜ୀ଴

The Most Probable Number is the value of n that maximizes this likelihood.

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Figure 14: Likelihood plots for a single dilution with fifty wells (top graph has seven positive wells, lower graph showing forty). The MPN is found by selecting the largest conditional likelihood. The plots demonstrate that the test is better at discriminating between small numbers of organisms - when many wells show growth the curve is broader.

5.3.1.4 Measures of Uncertainty As demonstrated in fig. 14, it is difficult to discriminate between MPNs when the majority of the wells show growth. An appreciation of the uncertainty introduced by the MPN technique is needed to design the test. This is much harder than determining the MPN and there is no exact way to calculate the uncertainty in the MPN result. In fact the confidence or credible intervals are subjective (McBride, 2003). One must make prior assumptions about the likelihood of contamination in order to determine a measure of uncertainty.

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Typically a diffuse prior (uniform distribution of mean bacterial densities) is used. Counter to experience and intuition, this assumption implies that high concentrations are more likely to be found than low concentrations. McBride (2003) suggests that water testing lends itself to the Bayesian statistics. He assumes the experimentalist is concerned with the probability of a particular result being within a range. While this may be the case for scientific studies, when tests are to be compared with risk assessments they are firmly in the classical statistical philosophy. The device is looking for performance in the long run rather than being specific about the current result. 5.3.1.6 Calculating Uncertainty The most traditional means of expressing uncertainty, the confidence interval, is not strictly appropriate for likelihoods and three alternatives were evaluated (Appendix 7.3). The Monte Carlo Method was suggested by Prof. Spiegelhalter as a means to determine the uncertainty of the measurement. Operational Characteristics: Monte Carlo From a risk profile, threshold values that the test should be able to discriminate between can be chosen. The values chosen are (based on examples in 2nd edition WHO Guidelines Vol.3 (1993) – pg. 78) are arbitrary: low risk (<10/100ml), medium (>50/100ml) and high risk (>100/100ml). A Monte Carlo method was developed to examine the operational characteristics of different device designs. It is described in the Appendix. This also facilitated the examination of thresholds in line with the chosen risk levels: to establish how many wells are needed to distinguish between chosen E.coli concentrations.

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Figure 15: Discrimination between threshold values using Monte Carlo – a comparison of 25 and 50 wells with fixed total sample volume of 50ml

Table 11: Discrimination between example threshold values for a 50ml sample

Number of Wells 5 10 20 25 50

Discrimination between 10 10 and 50/100ml and 100/100ml Good Good Good Good Good Poor Adequate Good Good Good None Poor Poor Adequate Good 50 and 100/100ml

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Findings and implications for the design 1. The device is looking for assured quality in a large number of different water supplies and thus requires a frequentist approach. We are not interested in the scientific measurement of the exact value of the MPN. The user is only interested in the extent of contamination of the water and the resultant health risk of drinking it. 2. The MPN method is not a precise technique. When many wells show growth, there is poor discrimination. 3. There is a trade-off between complexity of test (both design and interpretation) and precision. It is easier to interpret the device if all of the wells are the same size. 4. The first prototype does not appear to be adequate for the purposes required. 5. The high bacterial concentrations expected in source selection (often >1000/100ml) are a challenge for a device using MPN: the well sizes would have to be very small. It may be necessary to design two devices: one for assessing drinking water and treated supplies and the second for more contaminated supplies such as surface waters.

5.3.2 Energy Strategy
Purpose: to ensure bacterial growth and reliable results within a reasonable time. In Section 5.1.1 it was found that the temperature must remain within the range 23 to 44°C. Growth is less sensitive than the precision of electronic incubators first led the author to believe. To maintain temperatures close to the optimal and within this range, several approaches were considered. These can broadly be termed low-tech and high-tech. The options for storing and accepting heat that were considered in this project are described below (fig. 16). Temperatures were recorded using an Omega 4-channel Datalogger in an environment fluctuating between 16 and 20°C. Whilst these do not reflect tropical temperatures or diurnal variations, the results give a good indication of the approaches that are likely to be successful.

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Figure 16: Heating and heat storage options that were considered
Chemical Energy Exothermic Reaction Insulated water bath Liquid-Gas Vapourisation Solid-Liquid Melting Latent Heat Solid-Gas Sublimation Solar Solid-Solid Accept energy Biomass Organic Phase Change Materials Lauric Acid T=42-44 1-Tetradecanol T=38 Inorganic PCM Salt Hydrates

Sensible Heat Store Energy

Body Heat

5.3.2.2 “Low-tech”: Body Heat Body surface temperature is typically between 32 and 35°C, higher on some areas of the body such as the armpits. It was soon realized that placing the device on the body gave sufficient temperature regulation, a finding validated by experiments (fig.16). Body heat has been shown to be a very effective solution.
Figure 17: Incubation using body heat (50ml sample)

35 30 Temperature (°C) 25 20 15 10 5 0 0 0.5 1 Time (hours) 1.5 2 2.5 3 Water Temperature Environment Temperature

Whilst acceptability can only be established through field trials, reports from South Africa suggest that individuals and households will be happy to carry the device with them for the duration of incubation (Gundry, 2008). For these users, where there is a choice between speed of the assay and cost, the design should invariably aim for the lowest cost. Aquatest: An Affordable Water Test | 5 Part II: Constructive Research 35

The use of body heat however is unlikely to be accepted by water professionals. They will not want to have several water samples attached to their bodies all day and night. It is suggested that high-tech solutions may have a role for incubating several samples at a time or during transport to a location where laboratory incubation is available. 5.3.2.3 A Reusable Incubator? A simple solution which could also solve the temperature requirement for professional users is to heat water to just above the optimal temperature and warm the devices in this water. The correct temperature could be attained by using a thermometer, or mixing three parts boiling water and one part source water. The samples could then be stored in an insulated container (e.g. vacuum flask) with a fraction of the warm water. This approach was evaluated using a 0.5l thermos flask. After 33 hours the temperature had dropped from 42°C to 24°C, within the required limits. The main problem with this approach is the need to boil and transport more water. The viability of Phase Change Material (PCM) to incubate several water samples (taken as 0.5l of water roughly ten) at a time was assessed through a combination of experiments and (lumped mass) heat transfer modelling. This began with a test of Portatherm - the incubator used in the MIT kit. It was unable to maintain a steady temperature despite containing only 500ml of water and over 2kg of PCM: either the insulation or the mass of PCM would need to be increased. Improving insulation is expected to be more cost effective and would result in a lighter incubator. 1-Tetradecanol, the PCM used in Portatherm, should not be ingested and its use is not recommended for safety reasons (note: a leading cause of poisoning in some developing countries is ingestion of kerosene). An alternative, Lauric Acid (Tm=42°C), was found by searching through two reviews of PCMs (Farid (2003), Zalba (2002)) and the CRC handbook.

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Findings and Implications for the Design

(i)

Body heat is by far the most appropriate solution. The device ought to be designed so that it can be kept near the body comfortably for over 36 hours.

(ii)

Phase change materials are not a viable option for a single sample and even less for a single-use device.

(iii)

Placing the device against the skin may not protect it from extremely high temperatures. In this case the device could be submersed in more water from the source and left in the shade.

(iv)

A reusable incubator would be desirable for professional users who need to undertake frequent monitoring. This incubator could use phase change in an insulated box – Lauric Acid is recommended for this purpose.

5.3.3 Bubbles and Stability
Purpose: response to problem during prototyping of narrow tubes 3mm I/D. Narrow blocked tubes would not fill when submerged in source water. When a narrow tube blocked at one end is submerged, air cannot escape and a bubble chamber forms. Surface tension forces dominate and water is stable above air. As the radius is increased the gravitational forces become more important and at a certain value, the bubble is no longer stable and the tube fills with water. This radius, ‫ݎ‬௖௥௜௧ , had to be determined because it would impact the form of potential designs, if not the minimum volume of water that could be separated. A stability analysis can be used to describe this phenomenon, the result of which gave an upper bound on the critical radius as just less than 5mm. Mestrel (2008) gives a derivation of the critical radius, leading to Equ. 2:

ሺ࢑࢘ࢉ࢘࢏࢚ ሻ࢓࢏࢔ ૛ >

ࢍ࢘ࢉ࢘࢏࢚ ૛ ሺ࣋૚ ି࣋૛ ሻ ࢽ

Equ. 2

Where ሺ݇‫ݎ‬௖௥௜௧ ሻ௠௜௡ is the smallest value given by the Bessel function which satisfies the rigid wall boundary condition J’m(kr) = 0. Figure 17 demonstrates that this is on the (blue) J1 (curve); it has a value of 18.41. This predicts an antisymmetric perturbation as is to be expected: air must go up one way as the water goes down the other.

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The numerical result in these lecture notes (r = 2cm) was in fact correct. The answer above was confirmed by a second source (Joseph, 1970) and experiments were conducted to verify this model.

Figure 18: Bessel Function

Kitchen experiments This was a very simple experiment: holes of different diameters were drilled into an acrylic block. The block was then carefully submerged in dyed water. Holes larger than the critical radius filled with the dyed water: the critical radius was between the smallest tube that filled and the largest tube that remained empty (fig 19).

Figure 20: Experiments to determine critical radius

Figure 19: Equivalence between empty submerged tube and suspended filled tube

Spurious initial results could be accounted for by residual soap in the water (as confirmed by later experiments). It is useful to note that impurities (e.g. turbidity) will always weaken the surface tension, which supports the use of the surface tension of pure water in determining the upper bound. Eq. 1 predicts that the stability depends only on density and surface tension. Results supported this theory for deep wells (40 mm), but nearly all of the 10-15mm deep wells filled. This suggests that the stability also depends on the depth of the well. The theory was Aquatest: An Affordable Water Test | 5 Part II: Constructive Research 38

also verified by an equivalence it implies (fig.20) between this problem and the emptying of narrow tubes filled with water (then blocked at one end).

Findings and Implications for the Design • The trapping of air in thin wells is a limit on the smallest volume of water that can be divided by pouring water into or submerging the device. This is therefore a limit on the range of the MPNS method. One way around this is to pipette the water into the device. • • The diameter should be at least 10mm to ensure that the wells can be filled by submersion in water so that bubbles will not be stable. By inspection, the smallest wells in the first Aquatest prototype will suffer from this problem.

5.4 Concepts and Their Evaluation
Table 12: Morphological Chart

Sample
Directly into device e.g. Bailer Sample cup/whirlbag Transfer using disposable pipette

Reagent
Pill packet Powered Saturated paper Hydrated

Incubate
Body Heat Organic PCM + insulate Body heat + insulate Exothermic reaction Mixing boiling water

Make safe
Solar water disinfection Chlorine disinfection Boil

Dispose
Sterilise in lab Sterilise in field Generate waste Recycle Biodegradable

A morphological chart (Tab. 12) was used to explore different combinations of functions. Having established that body heat would adequately incubate the water sample, the critical function was taking the sample. With indicator bacteria present in the environment, it would need to be very simple in order to make sure it is carried out aseptically. The most promising concept is described on the next page; other ideas are given in the Appendix 7.4.

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Final Concept: : pipette transfer into the device The Aquatest prototype would wou be complicated to use and relatively wasteful. It would use gloves and the device would be packaged in a sterile bag. The risk of contaminating these when opening the bag would be high. A much simpler solution is to use a sterile disposable pipette to transfer ransfer water from the source to the device. As pipettes are operated away from the water, hands can be kept clear from the device. Using a pipette also avoids the bubble stability limit described in section 5.3.3. 5.3.3 A screw-on on lid with neoprene under it is suggested to stop cross-contamination contamination of the wells. The neoprene could be white e as shown in the figure to facilitate reading the results. The lid (shown in green) would need to be opaque to prevent unintentional denaturisation of E.coli due to solar disinfection, but it is suggested that the colour is not dark as this may lead to overheating. The screw-on on lid should be deep, entirely encasing the sides of the base, so that it is not easy to contaminate the interior. The base (shown in grey) must be translucent trans and would preferably be transparent.

Figure 23: Pipettes or sterile Figure Figure 21: Final 22: Final concept Concept waterjet Waterjet demonstration model syringe demonstration model

Figure 22: : Final design concept c CAD drawing

The choice of materials will be determined on whether the device is expected to be reused, and if so where the re-sterilisation sterilisation will take place. Generating waste at the point of use is not to be encouraged both because the collecting systems are not available and because this would impose the western “disposable culture”. However, the author recognises that it may be difficult to implement a collection scheme and furthermore furthermore this may result in a further dependence on centralised laboratories. Aquatest: An Affordable Water Test | 5 Part II: Constructive Research 40

If the device is to be reused, the lid could be made from polypropylene and the base from polystyrene, both of which are resistant to UV and can be autoclaved. A disposable device would need to have a seal to show that the device has not been used.

6 Final Conclusions
6.1 Findings
Water testing 1. Without stronger epidemiological evidence quantitative water quality testing is only a measure of the extent of faecal contamination: it does not measure health risk. 2. An affordable water test can be produced using the MPNS method. This will be much more suited to the needs of the rural poor than current equipment. 3. The significance of water quality and in particular the health benefits of improving the quality of water supplies have often been overstated. In spite of water quality being important it is also essential to have an integrated approach to solving health problems. Design of the Aquatest device 1. Field testing kits were designed to strict specifications – many of these are not justified in this context and add unnecessary expense. Minimum requirements for the device have been proposed. 2. Aquatest ought to pursue “low-tech” incubation methods: the use of body heat is deemed appropriate. 3. Statistical models suggest that ten wells may not provide enough information to distinguish between sources. A statistical model has been developed which will enable the configuration to be determined once the thresholds are set. 4. Care must be taken when using small wells (r<5mm) as these may not fill with water when submerged. 5. The first prototype is complicated and can be simplified considerably in order to make it easier to use and less expensive to manufacture.

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6.2 Recommendations and Further Work
The project has uncovered many areas that need to be addressed by the Aquatest Initiative. The most important of these are summarised below: 1. Stronger epidemiological evidence is needed to support the use of E.coli as an index. 2. The configuration of wells needs to be re-evaluated, once thresholds have been determined from the epidemiological evidence in (1). 3. Further work on ways to incubate samples for professional users is needed. 4. Acceptability of body heat to incubate samples needs to be verified. 5. The assumption of random distribution of E.coli in a sample needs to be validated. Mixing may be required as part of the sampling procedure to ensure that it is representative.

6.3 Critical Evaluation of the Project
The breadth of this project meant that some lines of inquiry could not be pursued to a completely satisfactory depth. Taking as a benchmark a statement by Eishenhardt Part I requires further exploration: ‘A study arrives at closure when theoretical saturation is reached: the point where the addition of further cases or further data would give rise to only incremental learning or improvement in the theory’ (Eishenhardt, 1989) Whilst the author felt, during the first half of the project, that it was inevitably limited by being an individual research project – it was this independence during the exploratory stages that led to some of the most interesting findings. The second half of the project would still benefit from further development time, but it is felt that the objectives were met: minimum requirements have been defined and new and useful ideas have been generated for the Aquatest device. This project forms a good basis from which the author proposes to participate in the Aquatest Initiative over the coming year.

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7 Appendix
7.1 Acknowledgements This project would never have been realised if it wasn’t for Amy Smith and her D-lab: thank you for putting some sense into my head. The project’s diverse nature has led me to interact with a broad range of people from a variety of background. I therefore would like to thank Tim Love, for helping me overcome my fears of Matlab, Prof. Spiegelhalter for indulging the statistical interests of an engineer and Prof. Sandy Cairncross at the London School of Tropical Health and Medicine for forcing me to question water testing yet again. Special thanks go to Prof. Peter Guthrie for bearing with my project as I led it further and further from his interests, to Hayley Sharp for leading me to Aquatest, and lastly to Dr. Gundry at the University of Bristol whose enthusiasm has been a driving force behind the project; I look forward to continuing the work with him next year.

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7.2 Case Study findings
Table 13: Case Study Findings

Research Finding
There are alternative means of monitoring water quality: e.g geological and sanitary surveys If health outcomes are the reason for testing then why not look at medical records if they are available? Fixed-site water testing can be inappropriate and limit the value of water testing programs

Justification
The cleanliness of the water was justified by the type of source The cleanliness of the water was justified by the improvements in the health of the community Testing undertaken by KHT was limited by the distance between Chiang Mai University laboratories and the rural villages: travel in developing regions is often difficult.

Action
Explore monitoring water quality by source Explore monitoring by outcome

Karen Hilltribes Trust

Compare portable water testing kits

According to the head of the charity, 'The Water testing can be culturally inappropriate Karen would have to be told to do the tests' because they would not see it is useful.

Explore Scientific-traditional and disease causation/transmission

The petridishes have a strong visual impact. Showing the results of the water testing to the Preference for MF over P/A The membrane filtration method is of more local community, demonstrated the visual and tests educational use than P/A tests. educational impact of the tests.

Escola Bradesco, Brazil

Testing should be appropriate to the Testing is only worthwhile if the result can be Explore different purposes of interventions that are or could be made. acted upon: e.g. water treatment, behavioural monitoring water quality Special regard should be given to the level of change or advocacy. accuracy required. There was a big difference in the reaction between the indian village and the portuguese Supports the need to examine settlements. The former did not want to hear Water testing can be culturally inappropriate scientific-traditional about the results, whilst the latter were education excited about having their water tested and getting the results back. Difficulty of getting consumables in country and their expense The consumables used in the MIT kit are difficult to find in many locations. Millipore's Cost Analysis of consumables m-ColiBlue24 broth is prohibitively expensive

Vigyam Ashram, India

The electricity supply in Pabal is not reliable Explore reliability of electrical Electricity supply can be unreliable, portable and the multiple tube method cannot be used grids worldwide AND incubators may be of use even if there is grid because there is roughly 6 hours a day without compare storage devices connection electricity. against incubator Current testing cannot distinguish between There is a need to discriminate between animal and human faecal contamination. I was sources of biological contamination, namely asked how this could be done so that the between animal and human faecal source of the contamination could be contamination. identified Need to keep costs of water testing equipment to a minimum Water testing being used for advocacy It may be necessary to dispel myths The project is being self-funded There is a need to pursuade the chief of the village that there is a problem with the water supply. The villagers believe that rainwater is not clean - and is "poison" Explore techniques for discriminating between sources of contamination AND findout when this is important Cost Analysis of current water testing kits Explore simple ways of explaining the results Explore scientific vs. traditional

Cross-case Analysis highlighted the need to investigate scientific vs. traditional education. Testing may encounter resistance and may be misinterpreted. There are many ways of “measuring” water quality and these needed to be explored.

Student Project, Indonesia

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7.3 Statistics Appendix
Table 14: Measures of uncertainty

Name

Description
Assuming the likelihoods are Poisson

Source

Findings
It is not easy to compare

Coefficient of Variation

results to determine distributed, the coefficient of variation can be derived over the range. This is the variance divided by the mean value. thresholds All likelihoods that are at least 95% of the Tillet likelihood of the MPN lie within the Most means of defining an (1995) Probable Range Uses Monte Carlo methods to determine the tell if the device is able to distribution of MPNs that the device would Author discriminate between give for a given concentration in the sample. concentrations. interval for likelihoods. The method allows you to This is strictly the only Halvorson whether the test can (1932) distinguish between

Most Probable Range

Operational Characteristics

Coefficient of Variation The graph (fig. 24) was evaluated using a measure of the coefficient of variation derived by Halvorson (1932:336). This method is useful in order to get an appreciation of how the number of wells determines the precision and the range of the device.
Figure 24: Graph of Coefficient of Variation

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Most Probable Range

Figure 25: Plots demonstrating the Most Probable Range

The most probable range is relatively straightforward to calculate – it is the range within which all likelihoods are greater than 95% of the likelihood of the MPN (as demonstrated in the figures above). This is useful for tabulating an uncertainty range for any given MPN, but does not give an indication of how the device would perform for a given test. For this reason the Monte Carlo method was developed. Monte Carlo Method For a given threshold density and total volume the number of E.coli is determined. This many random numbers were allocated into categories as shown in the image (fig. 26) mimicking random distribution of E.coli in water. The total number of categories with at least one number gave the number of tubes showing growth. The MPN could then be found from tables (calculated This process using was
Figure 26: Random number allocation

Occupancy

theory).

repeated 10,000 times. Aquatest: An Affordable Water Test | 7 Appendix 46

7.4 Concept Generation Figures start at 27, Tables at 15

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7.5 Risk Assessment Retrospective Before the prototyping started a comprehensive risk assessment form was filled in along with COSH forms for 1-Tetradecanol and Lauric Acid (the Phase Change Materials used in this project). The process of completing the COSH forms not only made me aware of the correct procedures, but also alerted me to the fact that 1-Tetradecanol is not safe to ingest and for safety reasons should not be used in the testing device. The main risk however was identified as using a hot air gun to join thin film plastic sheets (note: not mentioned in report). 7.6 References Beliaeff, B. M.-V. (1993). The "Most Probable Number" Estimate and its Confidence Intervals. Water Research , 799-805. Blum D, F. R. (1983). Measuring the impact of water supply and sanitation investments on diarrhoeal diseases: problems in methodology. International Journal of Epidemiology , 357-65. Cairncross, S. F. (1993). Environmental Health Engineering in the Tropics: an introductory text 2nd Edition. Chichester: John Wiley. Centre for Disease Control and Prevention. (2008). Health Information for International Travel. Atlanta, USA: Elsevier. Clarkson, J. C. (2007). Inclusive Design Toolkit. Cambridge: Engineering Design Centre. Clasen, T. C. (2007). Interventions to improve water quality for preventing diarrhoea: systematic review and metaanalysis. British Medical Journal , 782-791. CRC. (1995). CRC handbook of chemistry and physics : a ready-reference book of chemical and physical data. Boca Raton, FL, USA: CRC. Cross, N. (1994). Engineering Design Methods: Strategies for Product Design 2nd Edition. Chichester: Wiley. David, F. N. (1962). Combinatorial Chance. London: C. Griffin. Davis, A. P., & Gundry, S. (2007). AQUATEST OVERVIEW: A study to develop low cost, accessible and affordable tests for fecal contamination of water in the developing world. DelAgua. (2008). DelAgua Quote dated 03/01/08. Marlborough. Eishenhardt, K. (1989). Building theories from case study research. Academy of Management Review , 532-550. Farid, M. K.-H. (2003). A review on phase change energy storage materials and applications . Energy Conversion and Management , 1597-1615. Feachem, R. G. (1978). Water, Health and Development: an interdisciplinary evaluation. London: Tri-Med Books Limited. Fewtrell L, K. R. (2005). Water, sanitation and hygiene interventions to reduce diarrhea in less developed countries: a systematic view and metanalysis. Lancet Journal of Infectious Diseases , 5:42-52. 48

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Fujioka, R. R. (1997). Sources of faecal indicator bacteria in a brackish, tropical stream and their impact on recreational water quality . Water Science and Technology , Vol. 35, 179-186. Garthright, W. E. (2003). FDA's preferred MPN methods for standard, large or unusual tests, with a spreadsheet . Food Microbiology , 439-445. Glass, R. I. (1997). Rotavirus vaccines at the threshold. Nature Medicine , 3: 10-11. Gleeson, C. G. (1997). The Coliform Index and Waterborne Disease: Problems of microbial drinking water assessment. London: E & FN Spon. Gordon, C. (2006). Low Cost Water Quality Testing for Developing Countries. CUED 4th Year Project. Gundry. (2008). personal communication. Gundry, S. W. (2006). Contamination of drinking water between source and pointof-use in rural households of South Africa and Zimbabwe: implications for monitoring the Millennium Development Goal for water. Water Practice & Technology , Vol. 1:2. Gupta, S. K. (2007). Usefulness of the hydrogen sulfide test for assessment of water quality in Bangladesh. Journal of Applied Microbiology , 388-395. Hakim, C. (1987). Research Design: strategies and choices in the design of social research. London: Unwin Hyman. Halvorson, H. O. (1933). Application of Statistics to Problems in Bacteriology II. Journal of Bacteriology , 331 - 339. Hofkes, E. H. (1983). Small Community Water Supplies: Technology of Small Water Supply Systems in Developing Countries. The Hague: Wiley.

Hunter, P. R. (1997). Waterborne Disease: Epidemiology and Ecology. Chichester: John Wiley. Hutton, L. (1983). Field Testing of Water in Developing Countries. Marlow: Water Research Centre. IWA. (2003). Assessing Microbial Safety of Drinking Water: Improving Approaches and Methods. IWA. J. Pillai, R. G. (1997). Bacteriological water testing by H2S method. 23rd WEDC Conference. Australia. Jensen, P. K. (2004). Is there an association between bacteriological drinking water quality and childhood diarrhoea in developing countries? Tropical Medicine and International Health , 1210–1215. JMP. (2006). Meeting the MDG Drinking Water and Sanitation Target: The Urban and Rural Challenge of the Decade. Geneva, Switzerland: WHO. Joseph, D. D. (1970). Stability of Fluid Motions II. Berlin: Springer-Verlag. Mara, D. D. (1973). Low-cost Facilities For the Bacteriological Examination of Drinking Water Samples. Water Research , 1243-1245. McBride. (2005). Using statistical Methods for Water Quality Management. Hoboken, NJ: Wiley. McBride, G. (2003). Uncertainty in Most Probable Number Calculations for Microbiological Assays. Journal of AOAC International , 1085-1088. Moe CL, S. M. (1991). Bacterial indicators of risk of diarrhoeal disease from drinkingwater in the Philippines. Bulletin of the World Health Organisation , 305-17. Polak, P. (2008). Out of Poverty: when traditional approaches fail. 49

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Prüss, A. K. (2002). Estimating the Burden of Disease from Water, Sanitation and Hygiene at a Global Level. Environmental Health Perspectives , 110(5): 537–542. Schumacher. (1973). Small is Beautiful: a study of economics as if people mattered. London: Blond and Briggs. Shefelbine, S. C. (2002). Good Design Practice for Medical Devices and Equipment: Requirements Capture. Cambridge: Engineering Design Centre. Thompson, J. (2001). Drawers of Water II: 30 years of change in domestic water use & environmental health in East Africa. London: International Institute for Environment and Development. Tillet, H. E. (1995). Comment on: "The 'most probable number' estimates and the usefulness of confidence intervals" by Beliaeff, B. and Mary, Y., J. Water Research. 27, 799-805 (1993). Water Resources , 1213-1214. VanDerslice, J. B. (1993). All coliforms are not created equal: a comparison of the effects of water source and in-house contamination on infantile diarrheal disease. Water Resources Research , 1983–1995.

WHO. (2006). Drinking Water Guidelines 3rd Edition. WHO. (2002). Evaluation of the H2S Method for Detection of Fecal Contamination of Drinking Water. Geneva: WHO . WHO. (1997). Guidelines for DrinkingWater Quality Vol.3 2nd Edition . Geneva: WHO. WHO. (2004). Water, sanitation and hygiene links to health: Facts and Figures updated Nov 2004. Geneva, Switzerland: WHO. Wright, J. G. (2004). Household drinking water in developing countries: a systematic review of microbiological contamination between source and pointof-use. Zalba, B. M. (2002). Review on thermal energy storage with phase change: materials, heat transfer analysis and applications. Applied Thermal Engineering , 251-283. Zoeteman, B. C. (1980). Sensory Assessment of Water Quality. Oxford: Pergamon.

Web Resources IWP. (2007). http://www.indiawaterportal.org/data/kits/h2s.html UNICEF. (n.d.). http://www.childinfo.org/eddb/Diarrhoea/progress.htm WHO. (2008). http://www.who.int/water_sanitation_health/hygiene/envsan/sanitchallenge/ Mestel, J. (2008). http://www.ma.ic.ac.uk/~ajm8/Hydrostab/kelvin.pdf. Kelvin-Helmholtz, Rayleigh-Taylor and capillary instabilities. Aquatest: An Affordable Water Test | 7 Appendix 50