This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. Article Cite This: Environ. Sci. Technol. 2018, 52, 12089−12097 pubs.acs.org/est Do Sanitation Improvements Reduce Fecal Contamination of Water, Hands, Food, Soil, and Flies? Evidence from a Cluster-Randomized Controlled Trial in Rural Bangladesh Ayse Ercumen,*,†,‡ Amy J. Pickering,§ Laura H. Kwong,∥ Andrew Mertens,‡ Benjamin F. Arnold,‡ Jade Benjamin-Chung,‡ Alan E. Hubbard,‡ Mahfuja Alam,⊥ Debashis Sen,⊥ Sharmin Islam,⊥ Md. Zahidur Rahman,⊥ Craig Kullmann,# Claire Chase,# Rokeya Ahmed,∇ Sarker Masud Parvez,⊥ Leanne Unicomb,⊥ Mahbubur Rahman,⊥ Pavani K. Ram,○ Thomas Clasen,◆ Stephen P. Luby,¶ and John M. Colford, Jr.‡ See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. † Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, North Carolina 27695, United States ‡ School of Public Health, University of California, Berkeley, California 94720, United States § Civil and Environmental Engineering, Tufts University, Medford, Massachusetts 02153, United States Downloaded via 138.220.206.73 on March 5, 2019 at 21:59:16 (UTC). ∥ Civil and Environmental Engineering, Stanford University, Stanford, California 94305, United States ⊥ Infectious Disease Division, International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka, 1212, Bangladesh # Water Global Practice, World Bank, Washington, D.C. 20433, United States ∇ Water Global Practice, World Bank, Dhaka, 1207, Bangladesh ○ University at Buffalo, Buffalo, New York 14214, United States ◆ Rollins School of Public Health, Emory University, Atlanta, Georgia 30322, United States ¶ Infectious Diseases & Geographic Medicine, Stanford University, Stanford, California 94305, United States * S Supporting Information ABSTRACT: Sanitation improvements have had limited effectiveness in reducing the spread of fecal pathogens into the environment. We conducted environmental measurements within a randomized controlled trial in Bangladesh that imple- mented individual and combined water treatment, sanitation, handwashing (WSH) and nutrition interventions (WASH Benefits, NCT01590095). Following approximately 4 months of intervention, we enrolled households in the trial’s control, sanitation and combined WSH arms to assess whether sani- tation improvements, alone and coupled with water treatment and handwashing, reduce fecal contamination in the domestic environment. We quantified fecal indicator bacteria in samples of drinking and ambient waters, child hands, food given to young children, courtyard soil and flies. In the WSH arm, Escherichia coli prevalence in stored drinking water was reduced by 62% (prevalence ratio = 0.38 (0.32, 0.44)) and E. coli concentration by 1-log (Δlog10 = −0.88 (−1.01, −0.75)). The inter- ventions did not reduce E. coli along other sampled pathways. Ambient contamination remained high among intervention households. Potential reasons include noncommunity-level sanitation coverage, child open defecation, animal fecal sources, or naturalized E. coli in the environment. Future studies should explore potential threshold effects of different levels of community sanitation coverage on environmental contamination. ■ BACKGROUND Diarrheal disease, intestinal parasites, and subclinical enteric pathogens from the environment to new hosts while hand- washing also reduces person-to-person transmission.3 Water infections are transmitted through environmentally mediated treatment and handwashing have been shown to lower fecal pathways,1,2 which can be interrupted by water, sanitation and hygiene interventions. Sanitation as a “primary barrier” isolates Received: June 2, 2018 fecal matter from the environment to prevent the spread of fecal Revised: September 12, 2018 pathogens and reduce fly breeding sites. Water treatment and Accepted: September 26, 2018 handwashing as “secondary barriers” reduce the transmission of Published: September 26, 2018 © 2018 American Chemical Society 12089 DOI: 10.1021/acs.est.8b02988 Environ. Sci. Technol. 2018, 52, 12089−12097 Environmental Science & Technology Article contamination of drinking water and hands, respectively, and double-sized control arm which received no intervention. reduce reported diarrhea.4−7 In contrast, sanitation interven- Details of the trial design have been reported.23 The primary tions have shown mixed health impact and generally no effect on outcomes of WASH Benefits were child diarrhea and growth, domestic environmental contamination.8−11 Larger impact can and additional trial outcomes included protozoa and soil- potentially be achieved by augmenting sanitation improvements transmitted helminth infections; these have been reported with water treatment and handwashing to synergistically separately.24−26 Measures of environmental contamination were interrupt multiple disease transmission pathways. prespecified intermediate outcomes.23 Few studies have assessed how water, sanitation, and hygiene We conducted an environmental assessment among the interventions affect disease transmission through pathways control, sanitation and combined water, sanitation and other than the conventionally studied routes of drinking water handwashing (WSH) arms of the trial (see Supporting and hands.11 In low-income countries, fecal contamination is Information (SI) Text S1 and Figure S1 for details of all pervasive on surfaces and objects in the domestic environment12 environmental assessments nested within WASH Benefits). The and ambient waters used for bathing and washing dishes.13 Flies sanitation intervention included upgrades to concrete-lined carry fecal pathogens14,15 and can transmit these to stored double-pit latrines, and provision of child potties and scoops for food;16 fly control programs have successfully reduced diarrheal the disposal of human and animal feces. Households in diseases.17,18 Soil is increasingly recognized as a reservoir for Bangladesh are clustered in multifamily compounds. All fecal organisms and has been linked to fecal contamination of households in the compound where the enrolled women and drinking water, hands, and food;19 ingestion of soil by children their newborns lived received latrine upgrades, potties and has been associated with environmental enteric dysfunction and scoops in order to reduce fecal contamination in the shared stunting.20 Identifying which of these transmission pathways are compound environment. Enrolled compounds made up <10% blocked by different interventions elucidates the mechanisms of a given geographical area because of the eligibility criterion of through which water, sanitation and hygiene programs improve having a pregnant woman. As such, the trial did not provide health and allows broader understanding of how findings might community-wide sanitation coverage. The water treatment and generalize to other settings. Without measuring fecal contam- handwashing interventions targeted the household where the ination in different environmental matrices as a causal inter- enrolled women lived and included (1) point-of-use water mediate, trials are limited to a “black box” understanding, where treatment with sodium dichloroisocyanurate (NaDCC, Aqua- underlying mechanisms of interventions are unknown and inves- tabs) (Medentech, Wexford, Ireland) and safe storage in a tigators can only speculate about reasons for intervention suc- narrow-mouth, lidded container with spigot and (2) provision of cess or failure. handwashing stations in the kitchen and latrine areas with a We collected environmental measurements within a random- water reservoir, a bottle containing a soapy water mixture and a ized controlled trial in rural Bangladesh (WASH Benefits, basin to catch the rinsewater. ClinicalTrials.gov NCT01590095) to assess how sanitation Community health promoters hired from among local women improvements, alone and combined with water and handwash- and trained specifically for the study visited intervention hous- ing interventions, affect fecal contamination along a compre- eholds on average six times per month throughout the trial to hensive set of environmentally mediated pathways. provide intervention products for free, replenish supplies as ■ MATERIALS AND METHODS Study Design. The WASH Benefits trial was conducted in needed throughout the study period and encourage user adherence. Intervention adherence, measured objectively with unannounced spot-check and structured observations as an four districts (Gazipur, Kishoreganj, Mymensingh, Tangail) in independent investigation from the study activities reported central rural Bangladesh. Groundwater in parts of Bangladesh here, was high throughout the study.27 Households in the inter- contains high levels of naturally occurring arsenic and iron.21 vention arms retained the intervention hardware (e.g., latrines, Study districts were chosen to have low concentrations of these potties, safe storage container) but the supply of consumables groundwater chemicals because the trial’s chlorine-based (chlorine tablets, soapy water solution) was discontinued after microbiological water quality intervention was not designed to the end of the trial. Control households did not receive any remove arsenic and also because the chlorine demand exerted by interventions or health promoter visits during or after the trial. iron limits the effectiveness of chlorination.22 The areas were No compensation was offered for participation. Further details also chosen to not have any major water, sanitation and hygiene of the intervention packages, health promoter visits and user programs that would interfere with study activities. adherence are reported elsewhere.27−29 The parent WASH Benefits trial enrolled pregnant women Procedures. We conducted the environmental assessment with the objective of following their birth cohort (referred to as after roughly 4 months of intervention. In the three study arms “index children” hereinafter). Field staff screened the study area selected for the environmental assessment, field staff from the for eligible women and recorded their global positioning system International Centre for Diarrhoeal Disease Research, Bangladesh (GPS) coordinates. Neighboring groups of eight eligible women (icddr,b) trained specifically for this study conducted house- were grouped into clusters based on their coordinates. Cluster hold visits to assess indicators of household water, sanitation dimensions were chosen such that one field worker could visit all and hygiene practices through spot-check observations and a cluster participants in 1 day. A minimum 1 km buffer was enforced structured questionnaire. They collected samples of source and between clusters to minimize spillovers of intervention effects and/ stored drinking water, ambient water from ponds adjacent to or intervention messages between study arms. Every eight adjacent enrolled households and used for bathing and domestic chores, clusters formed a geographic block. An off-site investigator (BFA) hand rinses from index children, food given to young children, block-randomized clusters into study arms using a random num- courtyard soil and flies captured near the kitchen to quantify ber generator, providing geographically pair-matched random- fecal indicator bacteria in the domestic environment (Figure 1). ization. The trial arms included individual and combined water, They also enumerated and speciated synanthropic flies in the sanitation, handwashing and nutrition interventions and a kitchen area. 12090 DOI: 10.1021/acs.est.8b02988 Environ. Sci. Technol. 2018, 52, 12089−12097 Environmental Science & Technology Article prioritized sampling rice if available. Field staff scooped the food from the dish it was provided in into a 50 mL sterile tube using a sterile spoon. To sample soil, the respondent was asked to identify the outdoor area where young children had most recently spent time. Field workers marked a 30 × 30 cm area using a stencil sterilized with ethanol between each household and scraped the top layer of soil within the stencil into a Whirlpak using a sterile scoop; the area was scraped once vertically and once horizontally to collect approximately 50 g of soil. To enumerate and sample flies, field workers identified a suitable location in the kitchen area (away from the stove smoke, under a roof or protected from rain) and horizontally hung three 1.5-foot strips of nonbaited sticky fly tape. They returned to the household 3−6 h later to count the captured synanthropic flies Figure 1. Flowchart of participant enrollment and environmental and speciate them according to a visual identification chart.32 sampling scheme. C refers to the control arm, S to the individual They removed one fly from the center of the strip with the most sanitation arm and WSH to the combined water, sanitation and flies using sterile tweezers and placed it into a Whirlpak. Samples handwashing arm. were transported to the icddr,b field laboratory on ice at 2−8 °C. Water, Sanitation, and Hygiene Indicators. Field workers Sample Processing. Samples were analyzed in 100 mL ali- observed drinking water storage conditions and recorded user- quots using IDEXX Quantitray (IDEXX Laboratories, Maine, reported water treatment practices. In households reporting USA) within 12 h of collection. Tubewell and stored water was chlorination, they measured the free chlorine residual in stored analyzed undiluted. Pond samples were diluted 1:100 (1 mL of drinking water with a digital colorimeter (Hach Pocket sample +99 mL of distilled water) and hand rinses 1:2 (50 mL Colorimeter II). They inspected the compounds for latrine of sample +50 mL of distilled water). Food and soil were access, presence of an improved latrine as defined by the Joint homogenized with distilled water in a sterile blending bag Monitoring Programme for Water Supply and Sanitation,30 and (Interscience, Saint Nom, France) using a laboratory-scale food latrine functionality and condition. They recorded caregiver- processer (Interscience, Saint Nom, France). A 10 g food aliquot reported child defecation and child feces handling practices. was homogenized with 100 mL water; 10 mL of homogenate Field staff also examined caregiver and index child hands was then mixed with 90 mL of distilled water. A 20 g soil aliquot (fingernails, fingerpads, palms) for visible dirt as a proxy for was homogenized with 200 mL water; 1 mL of homogenate was handwashing behavior31 and checked for the presence of water mixed with 99 mL of distilled water, serially repeated twice, to and soap within six steps of the latrine and kitchen. Finally, they generate a final 100 mL aliquot. Additional 5 g aliquots of food observed food storage conditions such as whether the storage and soil were oven-dried overnight to determine moisture container was covered or elevated to assess if these were indi- content. Flies were homogenized with a pestle from outside the rectly affected by the interventions even though the intervention Whirlpak and mixed with 100 mL of distilled water; 1 mL of packages did not entail any materials and behavioral messages slurry was then mixed with 99 mL of distilled water. Colilert-18 for hygienic storage of food. media was added to samples, followed by incubation at 44.5 °C Sample Collection. Samples were collected using sterile for 18 h to enumerate the most probable number (MPN) of Whirlpak bags (Nasco Modesto, Salida, CA). Clean gloves were Escherichia coli and fecal/thermotolerant coliforms33 (per 100 mL worn while collecting pond, hand rinse, food and soil samples. for water samples, per 2 hands for child hands, per 1 dry gram for To sample tubewells, field workers removed fabric or other food and soil and per 1 fly for flies). MPN values were derived materials from the tubewell mouth and flushed the well by from the number of yellow and/or fluorescent wells on the trays pumping five times before collecting 250 mL of water. To collect using the IDEXX Quantitray-2000 MPN table. Trays exceeding stored water, field workers asked the respondent to provide a the upper detection limit of 2419 MPN were classified as too glass of drinking water from the primary storage container in the numerous to count (TNTC); the Quantitray-2000 system with same manner they would give it to their children and pour this high detection limit was chosen to capture a range of approximately 150 mL into the Whirlpak. If the respondent contamination levels (see SI Table S1 for detection limits for reported using chlorine, sodium thiosulfate was used to each sample type). neutralize residual chlorine. Ponds were sampled by dipping a Quality Control. One laboratory control per analyst per day Whirlpak into the pond and collecting 250 mL of water from the and 5% replicates (repeat aliquots from same Whirlpak for every pond surface in the area where the household reported most 20th sample) were processed. Field workers collected 10% field commonly accessing the pond. Hand rinse samples were blanks (one blank for every 10 samples) by asking respondents collected from the index child (aged 1−14 months), and if not to pour distilled water from a sterile bottle into a Whirlpak as if available, from the next youngest child in the household. collecting a stored water sample and by opening and massaging a To sample child hands, field workers asked the respondent to prefilled Whirlpak as if sampling a hand. place the child’s left-hand into a Whirlpak prefilled with 250 mL Ethics. Participants provided written informed consent in the of distilled water. The hand was massaged from outside the bag local language (Bengali). The study protocol was approved by for 15 s and shaken for 15 s. The procedure was repeated with human subjects committees at the icddr,b (PR-11063), Univer- the right-hand in the same bag, and the rinsewater was preserved sity of California, Berkeley (2011−09−3652), and Stanford Uni- in the Whirlpak. To sample food given to young children, field versity (25863). workers identified stored food prepared to be served to children Statistical Methods. Our prespecified analysis plan is <5 years and asked the respondent to provide a small amount in registered and publicly available at Open Science Framework the same manner they would serve it to their children. They (https://osf.io/6u7cn/). 12091 DOI: 10.1021/acs.est.8b02988 Environ. Sci. Technol. 2018, 52, 12089−12097 Environmental Science & Technology Article Sample Size. The environmental assessment targeted all promotion was ongoing. Among households in the combined households enrolled in one of the double-sized control arms, WSH and sanitation arms, 94% had received a promotion visit sanitation arm and WSH arm of WASH Benefits for a total of within the last week, and 98% had received a visit within the last 2160 households (720/arm). We obtained measures of contam- 2 weeks. ination and intraclass correlation coefficients from the literature Water, Sanitation, and Hygiene Indicators. Drinking and unpublished pilot data (see analysis plan). We assumed the Water. Water treatment and safe storage practices were sub- interventions would decrease but not increase contamination4,6 stantially improved in the WSH arm (Table 1), with 95% of and therefore used a one-sided α of 0.05. Our sample size had 80% power to detect a 25% relative reduction in E. coli Table 1. Indicators of Water Treatment, Sanitation and Hand prevalence in tubewells and 0.20 log10 reduction in E. coli counts and Food Hygiene in stored water, in soil and on hands, compared to controls. Parameters of Interest. Our outcomes were (1) prevalence percent of households control sanitation WSHa and concentration of E. coli and fecal coliforms, (2) prevalence water storage container covered 28.4 29.0 95.3 and number of flies near the kitchen, and (3) prevalence of reported treating drinking water 1.8 1.9 92.0 caregiver and child hands with visible dirt. Our parameters were detectable (>0.1 mg/L) chlorine residualb 77.7 prevalence ratios (PR) and differences (PD) for the binary on-site latrine present in compound 97.2 100.0 99.8 measures, log10 reductions for E. coli and fecal coliform concen- primary latrine is improved latrine 67.4 96.0 98.5 trations, and fly count ratios for the number of flies. We primary latrine has functional water seal 36.1 93.1 94.4 substituted bacterial counts with half the lower detection limit primary latrine drains into environment 22.2 3.6 1.8 for nondetects and with 2420 MPN for TNTC samples.34 young children reported to defecate in potty/ 7.9 17.5 17.8 latrine Estimation Strategy. We compared both intervention arms child feces disposed of in latrine 9.7 29.8 30.9 to controls and the combined WSH arm to the single sanitation reported using scoopc to handle child feces 7.9 8.5 7.6 arm. Analyses were intention-to-treat; this preserves the ran- reported using scoopc to handle animal feces 34.7 64.6 63.8 domization and is appropriate given the trial’s high user water and soap available <6 steps from latrine 7.5 7.4 45.3 adherence to interventions.27 Randomization balanced cova- water and soap available <6 steps from kitchen 4.4 2.8 69.6 riates across arms.24 Therefore, we relied on unadjusted esti- food storage container covered 85.1 82.7 85.8 mates in our analysis. We estimated unadjusted parameters food stored at safe location (elevated/inside 72.0 69.8 74.4 using generalized linear models with robust standard errors, and cabinet) a log link for PRs, linear link for PDs and log10 reductions, and ≥1 fly caught in kitchen 31.9 34.0 35.3 log link allowing for overdispersion (negative binomial regres- % of houseflies among flies caught 85.2 96.5 88.7 sion) for fly count ratios. Secondary analyses adjusted for a WSH: Water, sanitation and handwashing. bAmong households prespecified covariates using doubly robust targeted maximum reporting having used chlorine to treat their stored drinking water. likelihood estimation (TMLE) incorporating an ensemble mach- c Sani-scoop provided by WASH Benefits or other feces removal tool ine learning method called Super Learner.35,36 We included an (e.g., garden hoe). interaction term for wet vs dry season to assess effect modification by weather conditions; Bangladesh has a pronounced monsoon drinking water storage containers fully covered (vs <30% in season from June through October which delivers >80% of the control and sanitation arms) and 92% of respondents reporting annual rainfall,37 and the wet conditions are typically associated treating their water (vs <2% in control and sanitation arms) at with higher levels of environmental contamination.19 ■ the time of our household visit. Reported water treatment in the WSH arm was exclusively by Aquatabs, and 78% of households RESULTS AND DISCUSSION reporting using Aquatabs had detectable (>0.1 mg/L) free Enrollment. Of 2098 households enrolled in the control, chlorine residual in their stored drinking water. sanitation and combined WSH arms of WASH Benefits, we Sanitation. Almost all control households had access to some enrolled 1840 (88%) in the environmental assessment between form of on-site sanitation facility (Table 1). Households in the July 2013 and March 2014. Reasons for households not being sanitation and WSH arms had substantially increased access to enrolled included no live birth or index child death (n = 158, improved latrines with hygienic isolation of feces, with >90% of 7.5%), absence or relocation (n = 72, 3.4%), refusal (n = 21, latrines having a functional water seal (vs 36% among controls) 1.0%) and no intervention hardware yet having been delivered and <5% draining into the environment (vs 22% among controls). (n = 7, 0.3%). Covariates were balanced between arms among Child feces management remained poor; in the sanitation and enrolled households (SI Table S2). WSH arms, < 20% of young children were reported to defecate Time Since Intervention Delivery. Political instability in in a latrine or potty, approximately 30% of respondents reported Bangladesh during the study delayed latrine construction in disposing of child feces in a latrine and <10% reported using the some areas. As a result, there was a short time window (1−4 sani-scoop to handle child feces, while approximately 65% weeks) between latrine construction completion and our envi- reported using it to handle animal feces (Table 1). ronmental assessment in 20% of enrolled households. Of the Handwashing. Access to water and soap for handwashing rest, 55% of households received their latrine construction near the latrine and kitchen was substantially higher in the WSH 1−6 months before and 22% 6−12 months before our visit. On arm compared to the control and sanitation arms (Table 1). average, the time window between latrine construction and our Among caregivers and children in the control arm, approx- environmental assessment was 4 months. All other hardware imately 50% of fingernails and 10% of fingerpads and palms had distribution (potties and sani-scoops, handwashing stations, visible dirt (Figure 2). Caregivers in the WSH and sanitation water treatment tablets, safe storage containers) had been arms had borderline reductions in the prevalence of dirt on completed for the entire study population with the exception of fingerpads/palms but not nails compared to controls; the seven households at the time of our visit, and behavior prevalence of dirt on child hands was similar across arms (Figure 2). 12092 DOI: 10.1021/acs.est.8b02988 Environ. Sci. Technol. 2018, 52, 12089−12097 Environmental Science & Technology Article Figure 2. Prevalence of caregivers and children with visible dirt on hands. C refers to the control arm, S to the individual sanitation arm and WSH to the combined water, sanitation and handwashing arm. Food Hygiene. Food storage practices were comparable reduction in E. coli prevalence (prevalence = 29%, prevalence across arms, with the majority of the containers covered and ratio [PR] = 0.38 (0.32, 0.44), p < 0.001) and approximately stored at a safe location elevated from the ground or inside a 1-log reduction in E. coli concentration (Δlog10 = −0.88 (−1.01, cabinet (Table 1). Among controls, at least one fly was captured −0.75), p < 0.001); no other pathway in the WSH arm had in 32% of kitchens and the mean number of flies was 1.13 (range: reductions in E. coli (Figure 3, SI Table S3). Secondary analyses 1−68). The predominant fly species was the common housefly adjusting for confounders yielded similar results (SI Table S3). (Musca domestica) in all arms. There was no reduction in the Subgroup analyses suggested larger reductions in E. coli in stored prevalence and number of flies in the sanitation or WSH arms drinking water during the dry season; there were no other compared to controls (Figure 3). seasonal effects (SI Table S4). Fecal coliforms showed similar Fecal Contamination. Levels of Contamination. We ana- patterns (SI Table S5). lyzed a total of 9940 samples. Half (49%) of households had a Quality Control. Across sample types, 41% of samples were pond and a third (32%) had a fly caught for analysis. Among nondetect and 5% exceeded upper detection limits. Intraclass controls, we detected E. coli in 25% of tubewells, 77% of stored correlation between samples processed in replicate was 86%. water, 43% of child hands, 58% of children’s food, 97% of ponds, E. coli was detected in 1% of blanks, and the geometric mean 95% of courtyard soil and 50% of flies (Figure 3). The ambient E. coli count among positive blanks was 6 MPN. Repeating the environment, such as ponds and soil, had extremely high contam- analyses after removing the data from days with contaminated ination; geometric mean E. coli was >5000 MPN per 100 mL of blanks did not change findings (SI Table S6). pond water and >120 000 MPN per dry gram for soil. Implications. The sanitation intervention, alone or combined Intervention Effects. There was no difference in the preva- with water treatment and handwashing, did not reduce fecal lence or concentration of E. coli for any sample type between the contamination of ambient and groundwater sources, child hands, sanitation and control arms (Figure 3, SI Table S3). Comparing food, flies and soil in the domestic environment. Chlorination the WSH arm to controls, stored drinking water had a significant and safe storage reduced E. coli in stored drinking water, 12093 DOI: 10.1021/acs.est.8b02988 Environ. Sci. Technol. 2018, 52, 12089−12097 Environmental Science & Technology Article Figure 3. Prevalence and concentration of E. coli (in source and stored drinking water, child hand rinses, food given to young children, pond water, courtyard soil, flies) and prevalence and number of flies captured near kitchen after approximately 4 months of intervention. E. coli concentrations are reported in the log of the most probable number (MPN) per 100 mL for tubewell, stored water and pond samples, per two hands for child hand rinses, per dry gram for food and soil samples and per fly for fly samples. C refers to the control arm, S to the individual sanitation arm and WSH to the combined water, sanitation, and handwashing arm. consistent with prior evidence.4 Intervention households a latrine or potty. Child feces are often rinsed into ditches or were subject to high levels of contamination in the ambient bushes or left on the ground in rural Bangladeshi households;39 environment. 30% of our respondents reported disposing of child feces in a Our findings are consistent with previous sanitation trials that latrine and <10% reported using a scoop to handle child feces. generally found no impact on environmental contamination.8−10 Additionally, trial participants made up <10% of compounds These prior studies entailed community-level programs with low within the boundaries of study clusters because of the eligibility to moderate adherence; 38−65% of households in intervention criterion of having a pregnant woman. Enrolled compounds villages had indicators of improved sanitation vs 10−35% of were surrounded by nonstudy compounds whose sanitary controls.8−10 WASH Benefits Bangladesh implemented a conditions likely remained at baseline levels. In a separate compound-level intervention with upgrades to concrete-lined assessment in our study area, we found evidence for spillover of double-pit latrines for all households in enrolled compounds. environmental contamination between neighboring study and In the sanitation arm, > 90% of respondents had an improved nonstudy compounds.40 Our compound-level interventions latrine with a functional water seal, and structured observations were thus possibly not sufficient to impact the broader demonstrated high latrine use among adults.27 Low adherence environment without higher community-level coverage of is therefore unlikely to explain the lack of intervention effects improved sanitation. A growing body of evidence suggests that from sanitation. However, structured observations can over- community-level sanitation coverage may be more important estimate latrine use as an observer’s presence artificially than individual household sanitation for preventing child enhances socially desirable practices.38 Also, children continued diarrhea in rural settings.41,42 For example, in rural Mali, higher open defecation despite sanitation access and provision of child community-level sanitation coverage was more strongly potties; fewer than 20% of children were reported to defecate in associated with improved child growth and improved drinking 12094 DOI: 10.1021/acs.est.8b02988 Environ. Sci. Technol. 2018, 52, 12089−12097 Environmental Science & Technology Article water quality than individual household access to a toilet.43 possible that the sanitation intervention reduced human fecal Contamination from the community can enter the compound contamination but animal fecal contamination masked this on shoes and soles of compound residents or through surface effect. While two-thirds of participants in the sanitation and runoff and/or animal waste runoff. Bangladeshi families also use WSH arms reported using a scoop to dispose of animal feces, we soil from outside the compound to coat walls and courtyards. observed animal feces in 90% of study compounds (vs human Soil, in turn, can contaminate hands, stored water and food.19 feces in <5%), and the presence of domestic animals was Additionally, groundwater and ambient waters can receive associated with increased E. coli in soil, drinking water and stored subsurface infiltration from surrounding compounds. Studies in food.19 Testing a subset of 500 soil, stored water and hand rinse Bangladesh found that latrine density within 100 m of a well and samples from study households for molecular fecal markers within a pond’s catchment area is predictive of tubewell and revealed prevalent ruminant and avian markers while human pond water quality44,45 rather than the presence of a latrine markers were rare,56 highlighting the role of animal fecal sources. within the compound.46 However, a recent study showed that Our findings indicate high levels of ambient contamination infiltration from latrines is limited to a radius of a few meters47 not affected by sanitation improvements in this setting. This such that contamination from surrounding compounds through could be due to lack of community-level improved sanitation subsurface transport is unlikely to affect groundwater quality. coverage, child open defecation, animal fecal sources, or Nonetheless, wells can also receive surface contamination from naturalized E. coli. Studies measuring the impact of sanitation the surrounding environment through unsealed or malfunction- on contamination in the ambient environment should consider ing wellheads.48 alternative indicators to differentiate between human and animal Furthermore, almost all control households in our study fecal sources. Studies should explore if there are threshold effects population had some form of on-site sanitation, such that the associated with different levels of sanitation coverage and trial presented an improvement in latrine quality and drainage as different contrasts in pre- vs postintervention sanitation access well as access to tools for child feces management but not basic on environmental contamination. latrine access. A setting with lower latrine coverage in the control group and a larger contrast in sanitation access between controls and intervention recipients may be better poised to detect an ■ * ASSOCIATED CONTENT S Supporting Information impact on environmental contamination. The Supporting Information is available free of charge on the It is also possible that the window between intervention ACS Publications website at DOI: 10.1021/acs.est.8b02988. initiation and our measurements (4 months on average) was not enough time for water, sanitation and hygiene practices to Text S1. Environmental assessments nested within change or for environmental reservoirs to respond to any reduc- WASH Benefits (Figure S1). Types of samples collected for environmental assessments approximately 4 months, tions in fecal input. While the water quality improvements one year and two years after the initiation of WASH indicate that water treatment practices had taken hold, Benefits interventions (Table S1). E. coli detection units sanitation behaviors might not have changed sufficiently to and limits (Table S2). Enrollment characteristics by impact environmental contamination. Additionally, subsurface intervention group (Table S3). E. coli prevalence and infiltration from new pit latrines is higher during the initial few concentration measured in control, sanitation and WSH months and diminishes afterward, potentially due to the arms after four months of intervention (Table S4). formation of a layer (like the schmutzdecke on biosand filters) Subgroup analysis by dry vs wet season on E. coli that attenuates contamination.47 Finally, while E. coli typically prevalence (Table S5). Fecal coliform prevalence and survives <20 days in soil,49 it can persist for extended durations concentration measured in control, sanitation and WSH in tropical soils.50 It is therefore possible that a longer follow-up arms after four months of intervention (Table S6). E. coli period would capture more pronounced impact from sanitation prevalence and concentration (after data from dates with improvements on environmental contamination. contaminated blanks have been removed) (PDF) Despite the lack of reductions in environmental contami- nation, sanitation arm participants experienced reductions in diarrhea24 and infections with protozoa25 and soil-transmitted helminths,26 suggesting reduced fecal-oral pathogen trans- ■ AUTHOR INFORMATION Corresponding Author mission in this arm not reflected by our E. coli measurements. *E-mail: aercume@ncsu.edu. One explanation is that the impact of the sanitation intervention ORCID on fecal-sourced E. coli in the environment may have been Ayse Ercumen: 0000-0001-6002-1514 masked by “naturalized” E. coli since E. coli can be naturally pre- sent in tropical soils and waters.51 Naturalized E. coli are pheno- Notes The authors declare no competing financial interest. ■ typically identical to E. coli from fecal sources; the detection methods used in our study cannot distinguish between naturalized and fecal E. coli.52,53 The lack of E. coli reduction ACKNOWLEDGMENTS in soil, ponds, and groundwater could therefore be due to This research protocol was funded by the World Bank and by nonfecal E. coli in the environment. However, testing a subset of Global Development grant OPPGD759 from Bill & Melinda our soil samples with biochemical assays, phylogrouping and Gates Foundation to University of California, Berkeley to polymerase chain reaction (PCR) detection of genes associated International Centre for Diarrhoeal Disease Research, Bangla- with enteric vs environmental origin showed no differences desh (icddr,b, grant number 741). icddr,b acknowledges with between E. coli isolates in soil vs in fecal samples collected from gratitude the commitment of Bill & Melinda Gates Foundation animals and humans in the study area.54 and University of California, Berkeley to its research efforts. Another explanation is that fecal indicator bacteria cannot icddr,b is also grateful to the Governments of Bangladesh, differentiate between animal vs human fecal sources.55 It is Canada, Sweden and the UK for providing core/unrestricted 12095 DOI: 10.1021/acs.est.8b02988 Environ. Sci. 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