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Water quality at points-of-use in the Galapagos Islands
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International Journal of Hygiene and Environmental Health journal homepage: www.elsevier.com/locate/ijheh
Water quality at points-of-use in the Galapagos Islands William A. Gerhard a,∗ , Wan Suk Choi b , Kelly M Houck c , Jill R Stewart a a Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina, 170 Rosenau Hall, CB #7400, 135 Dauer Drive, 27599-7400 Chapel Hill, NC, USA b Department of Biostatistics, Gillings School of Global Public Health, University of North Carolina, 170 Rosenau Hall, CB #7400, 135 Dauer Drive, 27599-7400 Chapel Hill, NC, USA c Department of Anthropology, University of North Carolina, 301 Alumni Building, CB #3115, UNC-CH, 27599-3115 Chapel Hill, NC, USA
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Article history: Received 1 September 2016 Received in revised form 14 December 2016 Accepted 26 January 2017 Keywords: Escherichia coli Bacteroides Fecal coliforms Regrowth Post distribution contamination Microbial source tracking
a b s t r a c t Piped drinking water is often considered a gold standard for protecting public health but research is needed to explicitly evaluate the effect of centralized treatment systems on water quality in developing world settings. This study examined the effect of a new drinking water treatment plant (DWTP) on microbial drinking water quality at the point-of-use on San Cristobal Island, Galapagos using fecal indicator bacteria total coliforms and Escherichia coli. Samples were collected during six collection periods before and after operation of the DWTP began from the freshwater sources (n = 4), the finished water (n = 6), and 50 sites throughout the distribution system (n = 287). This study found that there was a significant decrease in contamination by total coliforms (two orders of magnitude) and E. coli (one order of magnitude) after DWTP operation began (p < 0.001). However, during at least one post-construction collection cycle, total coliforms and E. coli were still found at 66% and 28% of points-of-use (n = 50), respectively. During the final collection period, conventional methods were augmented with human-specific Bacteroides assays – validated herein – with the goal of elucidating possible microbial contamination sources. Results show that E. coli contamination was not predictive of contamination by human wastes and suggests that observed indicator bacteria contamination may have environmental origins. Together these findings highlight the necessity of a holistic approach to drinking water infrastructure improvements in order to deliver high quality water through to the point-of-use. © 2017 Elsevier GmbH. All rights reserved.
1. Introduction Diarrheal diseases are the sixth-leading cause of premature death worldwide, above tuberculosis, malaria, and traffic accidents (Lopez et al., 2006). Diarrhea can be caused by a range of viral, bacterial, and protozoan pathogens, which increases the complexity of addressing this problem (Fischer-Walker et al., 2013). The United Nations aimed to halve the proportion of the world population without sustainable access to drinking water between the years of 1990 and 2015 (United Nations, 2015). As a result, nearly two billion people gained access to piped drinking water (United
Abbreviations: DWTP, drinking water treatment plant; FIB, fecal indicator bacteria; LLOD, lower limit of detection; MPN, most probable number; MST, microbial source tracking; WHO, World Health Organization. ∗ Correspondence to: 170 Rosenau Hall, CB #7400, 135 Dauer Drive, 27599-7400 Chapel Hill, NC, USA. E-mail addresses: [email protected] (W.A. Gerhard), [email protected] (W.S. Choi), [email protected] (K.M. Houck), [email protected] (J.R. Stewart).
Nations, 2015). These improvements will no doubt improve public health. However, research is needed to explicitly evaluate the effect of piped drinking water on point-of-use water quality in developing world settings. It is possible for drinking water quality to deteriorate between treatment and the point-of-use, in which case the false presumption of safety may inadvertently increase the public health risk of water-borne disease (Bain et al., 2014; Rufener et al., 2010; Wright et al., 2004). For example, it has been reported that access to protected sources may reduce the rate at which consumers observe precautions such as boiling drinking water prior to consumption (Lindskog and Lindskog, 1988). As a result, improving access to drinking water without ensuring its quality may have unintended negative impacts on human health. This study examined point-of-use water quality on the Galapagos Islands of Ecuador, an area with piped drinking water that is also undergoing infrastructure improvements. Ecotourism to the islands has grown exponentially from 2000 tourists in the year 1960 to 120,000 tourists in 2005 (Epler, 2007). The popularity of ecotourism creates a framework called the Galapagos Paradox where increasing strain is placed on the “pristine” environment that many
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tourists hope to enjoy (Quiroga, 2009). Among other stressors, the strain that tourism places on the drinking water infrastructure has contributed to drinking water quality problems that have persisted for decades (Liu and D’Ozouville, 2013; Reyes et al., 2015). Effective drinking water infrastructure requires access to quality source water. On San Cristobal Island, the source water is from two freshwater lakes – La Toma de Los Americanos (Los Americanos) and Cerro Gato – in the highlands of the island. These two sources are fed by rainwater during the rainy season and have sufficient capacity to provide 30 L per second of water flow to the drinking water treatment plant (DWTP) year-round through the dry season. San Cristobal draws its drinking water entirely from these surface reservoirs rather than subsurface reservoirs like two of the other inhabited islands on the archipelago – Santa Cruz and Isabela. A project to replace the DWTP and distribution system on San Cristobal Island is underway. Construction of a new DWTP began in late 2012, and the new DWTP began operations in September 2013. This DWTP includes treatment processes common to modern drinking water treatment systems including rapid mixing, coagulation, flocculation, sedimentation, filtration, and chlorine gas disinfection. An infrastructure improvement project is ongoing as the drinking water distribution infrastructure is replaced; however, several problems that may allow for post-collection contamination continue to affect the distribution system. The distribution system is not consistently pressurized, instead operating as a gravity-fed system a few hours each day. Negative hydraulic pressure associated with interrupted service can draw contamination into water pipes from the surrounding environment (Lee and Schwab, 2005). Municipal sewer lines have also been laid immediately below the drinking water distribution system in the ongoing infrastructure improvement project. The vast majority (79%) of households are connected to municipal sewer lines for waste management while a smaller proportion (19%) of households uses septic tanks. The wastewater treatment plant on the island was not in operation during the time period of this study, and wastewater from the piped system was discharged directly to the ocean. For houses with septic tanks, the volcanic rock conditions likely prevent septic fields from functioning properly and may allow inadvertent contamination resulting from negative hydraulic pressure associated with inconsistent water delivery. Lack of consistent water availability necessitates the use of storage cisterns at points-of-use. Several studies have found increases in contamination during household storage and at least one study has identified the household level as the point at which the largest impact on public health can be made (Clasen and Bastable, 2003; Eshcol et al., 2009; Kær Jensen et al., 2002; Levy et al., 2008; Rufener et al., 2010; Wright et al., 2004). The cisterns on San Cristobal were above-ground plastic storage tanks. These cisterns are filled from the top by hoses that shutoff using a simple fill valve, so the opportunity for feedback into the distribution from cisterns is eliminated. Domestic animals are often near the cisterns on household property and may represent a possible route of contamination, but the likelihood of this contamination is reduced given the overwhelming majority of cisterns were covered. Variable cistern residence times also complicate the calculation of doses for residual disinfection of finished water. Despite the improvements made to the infrastructure, lack of consideration for the effects of intermittent water delivery and point-of-use water storage may allow bacteria in the environment to infiltrate the system and, under certain conditions, multiply. This study establishes baseline measurements of drinking water quality at various points of the distribution system on the island of San Cristobal over a two year period and provides the most comprehensive spatial representation of microbial drinking water quality on any island in the Galapagos archipelago to
date. Furthermore, this study examines the effect of a newly constructed drinking water treatment plant on microbial water quality, both in terms of comparisons between years and in terms of compliance with established World Health Organization (WHO) Guidelines. This study also evaluates the sources of observed fecal contamination through microbial source tracking (MST) methods. Because of spatially widespread microbial contamination observed during the first year of the study, human-specific assays were used to narrow the list of possible sources for microbial contamination. Several studies have applied this approach to drinking water source quality monitoring; however, few have applied it to drinking water qual¨ et al., 2015; ity monitoring through the distribution system (Åstrom Marti et al., 2013). This is one of the first studies to apply MST methods to examine drinking water quality at the point-of-use in South America.
2. Methods 2.1. Site selection The study area of San Cristobal Island includes the two main population centers: El Progreso (population of 500, altitude 700 m) and Puerto Baquerizo Moreno (population of 5400, altitude 0–70 m) (Fig. 1). This study also included the freshwater sources (Los Americanos and Cerro Gato) and the DWTP (altitude 700 m) – all located on San Cristobal. During June 2013, 50 sample sites from 15 neighborhoods on the island of San Cristobal were selected to represent locations throughout the drinking water distribution system. Each of these sites was also subject to post-distribution storage – typically in the form of rooftop or ground-level cisterns where water was stored prior to household use.
2.2. Sample collection A total of 143 point-of-use water samples from 50 sites along the distribution system were collected in three collection periods, which occurred in 7–10 day intervals in June and July 2013. A total of 144 samples from the same 50 sites were collected during three collection periods at the same intervals during the same months in 2014. Every effort was made to collect a sample from each site during each collection period, but inconsistent business hours and shifting schedules occasionally prevented collection from sites within 48 h of the start of a collection period. In these instances, the site was skipped for that collection period. The two freshwater sources for the island (Los Americanos and Cerro Gato) were included in the study to assess microbial contamination from the beginning to the end of the treatment and distribution processes. Construction of the new DWTP using a treatment process involving flocculation, sedimentation, rapid filtration, and chlorine-gas disinfection was completed in September 2013. Samples were collected weekly from the new DWTP in 2014. Water samples of 100 mL were collected in vacuum sealed, sterile 120 mL vessels containing sodium thiosulfate from all 50 sample collection sites during each collection period. The samples were placed in a cooler with ice packs and taken to the Galapagos Science Center for enumeration of total coliform and E. coli using ® the IDEXX Colilert method (Westbrook, ME USA) within 12 h of sample collection. Permit requirements limited the number of collection opportunities at the freshwater sources; however, there was one collection period in 2013 and three collection periods in 2014. Water (500 mL) was collected from each point-of-use during the final collection period in 2014 in double-washed and autoclaved glass laboratory bottles for MST analysis.
Please cite this article in press as: Gerhard, W.A., et al., Water quality at points-of-use in the Galapagos Islands. Int. J. Hyg. Environ. Health (2017), http://dx.doi.org/10.1016/j.ijheh.2017.01.010
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Fig. 1. Inhabited zones and neighborhoods of San Cristobal Island and neighborhoods of Puerto Baquerizo Moreno.
2.3. Total coliform and E. coli enumeration
2.4. Physical and chemical parameters
Total coliform and E. coli most probable number (MPN) per 100 mL were measured at the Galapagos Science Center ® using IDEXX Colilert (Westbrook, ME USA) according to the manufacturer’s protocol (Microbial Contaminants Method 9223, 2005).
All physical and chemical parameters were measured in 2014 using a YSI Professional Plus handheld multi-parameter instrument. Atmospheric pressure was measured using the internal barometer and temperature, dissolved oxygen, conductivity, and pH were all measured using the relevant YSI probe attachments.
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Recalibration of all probes was performed using external and internal standards before each collection period. Data were not collected in 2013 because appropriate instrumentation was not available on the island at that time. Chlorine residuals and water temperature in the distribution pipeline were not measured because direct access to the pipeline was not possible. In addition, there was not capacity to perform chlorine measurements in either year of the study. 2.5. DNA extraction and storage The 500 mL samples collected for MST analysis were filtered through 0.45 m polycarbonate filter paper using a vacuum apparatus. The resulting filter paper was stored in a −20 ◦ C freezer prior to transport to the University of North Carolina at Chapel Hill (UNC-CH) for DNA extraction and analysis. DNA extraction with performed in September 2014, two months after collection. DNA extraction was performed on all filtered samples using the ® MoBio PowerSoil DNA Isolation Kit (Carlsbad, CA USA) accord® ing to the manufacturer’s protocol. PowerSoil was chosen instead ® of PowerWater because prior studies show that the former can effectively prevent inhibition (Cox and Goodwin, 2013). In addition, studies have shown similar recovery with the two MoBio kits when using filter homogenized samples (Kaevska and Slana, 2015). The 100 L DNA extract was transferred into two single-use aliquots of 20 L and one reserve aliquot of 60 L which were stored at −80 ◦ C until they were examined with the PCR/qPCR assays beginning in February 2015, seven months after collection. 2.6. Conventional PCR Conventional PCR was performed at UNC-CH using the humanspecific HF183 Endpoint PCR assay as described by Bernhard and Field with slight modifications (Bernhard and Field, 2000). These modifications deviated from the initial study protocol primarily in the development of the master mix. The HF183 Endpoint assay was performed using the following master mix components (stock concentrations): HF183F (100 M), Bac708R (100 M), and 5 PRIME MasterMix (2.5×). The optimized final concentration of these reagents and the amount of reagent per reaction are found in Table S1. Forward and reverse primer sequences for this assay are described in Table S4.
at 60 ◦ C for 60 s, and a plate read; and (4) A melt curve of 60 ◦ C to 94.8 ◦ C, with steps of 0.4 ◦ C every 10 s. The HF183 TaqMan assay was performed using the following master mix components (stock concentrations): HF183F (100 M), BthetR1 (100 M), BthetP1 (100 M), and Fast Universal Master Mix (2×). The master mix was made in two steps: (1) Creating a primer/probe mixture from the stock primers/probes; and (2) Combining this prepared primer/probe mixture with the remaining master mix reagents. The optimized amount of reagent per reaction well is found below (Table S3). 2.8. HF183 prevalence Stool samples were collected from 22 residents of San Cristobal Island as part of an ongoing biological anthropology study. Informed consent was obtained, and the study protocol and analysis were approved by the Institutional Review Boards at the University of North Carolina at Chapel Hill and the Universidad San Francisco ® de Quito. Using the Omega Bio-Tek E.Z.N.A. Stool DNA Kit (Norcross, GA, USA), DNA was extracted from 200 mg of wet stool and eluted to a final volume of 50 L. These samples were analyzed using the conventional PCR and qPCR methods outlined above to determine the prevalence of human-specific Bacteroides in the local population. Six 100 mL samples were collected from the influent to the waste water treatment plant (WWTP) on two different dates (24 June 2014 and 18 July 2014) to examine the concentration of human-specific Bacteroides in raw sewage. Of these samples, 10 mL from each waste water sample was filtered and analyzed using the same DNA extraction protocol and PCR/qPCR assays as the drinking water samples. 2.9. DNA sequencing Positive point-of-use samples were confirmed via Sanger sequencing performed by Eton Biosciences Incorporated (Research Triangle Park, NC). The resulting DNA sequences were compared to known microbial genomes using the Basic Local Alignment Search Tool (BLASTN 2.2.31) available online through the National Center for Biotechnical Information (NCBI) (Zhang et al., 2000). The sequences were searched in the 16S ribosomal RNA sequences (Bacteria and Archaea) genome database.
2.7. Quantitative real-time PCR 2.10. Data analysis All quantitative real-time PCR (qPCR) was performed at UNC-CH using two human-specific HF183 assays (HF183 SYBR and HF183 TaqMan) (Haugland et al., 2010; Seurinck et al., 2005). Both assays were performed in a Bio-Rad CFX96 Touch Real-Time PCR Detection System (Hercules, CA, USA), which includes the Bio-Rad CFX96 Optical Reaction Module and the Bio-Rad C1000 Touch Thermal Cycler. The standard curve was calculated using DNA from Bacteroides dorei (GenBank reference AB242142) (Bernhard and Field, 2000). This DNA was serially diluted with copy number concentrations ranging from 108 per L to 101 per L. An eight-point standard curve using these concentrations in triplicate was generated for each run. The HF183 SYBR assay was performed using the following master mix components (stock concentrations): HF183F (100 M), HFsybR (100 M), dNTP with dUTP (5 mM), MgCl2 (50 mM), PCR 10× reaction buffer (10×), and Hot GoldStar DNA Polymerase (5 U/L). The optimized final concentration of these reagents and the amount of reagent per reaction well is found below (Table S2). The cycling conditions for HF183 SYBR were as follows: (1) 50 ◦ C for 2 min; (2) Polymerase activation at 95 ◦ C for 10 min; (3) 40 cycles of denaturation at 95 ◦ C for 30 s, annealing at 53 ◦ C for 60 s, extension
All microbial data (total coliforms and E. coli) was logarithmically transformed for data analysis. Microbial data below the lower limit of detection (1 MPN per 100 mL) were assigned the √ ) where LLOD represents the lower limit of detection. value ( LLOD 2
This method is widely used to generate fill-in values for samples below the LLOD when data are not highly skewed (Finkelstein and Verma, 2001). This method is appropriate for this study because the data have been log-transformed to better approximate normal distribution. Samples above the upper limit of quantification were assigned the value of this upper limit (2419.6 MPN per 100 mL). The arithmetic mean of the logarithmically transformed data was calculated for each site for 2013 and 2014. These values were compared between the years using a Wilcoxon Ranked Sum Test to establish the significance of differences. The hypotheses tested included: (1) H0 : T2013 = T2014 , where T2013 and T2014 imply arithmetic mean of ln(total coliforms) of all sites in 2013 and 2014, respectively; and (2) H0 : E2013 = E2014 , where E2013 and E2014 imply arithmetic mean of ln (E. coli) of all sites in 2013 and 2014, respectively. The hypothesis testing was performed using a 0.05 significance level (␣ = 0.05).
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A regression analysis was performed to determine if there was any relationship between microbial contaminant concentration and pH or water temperature. These analyses were performed with the SAS 9.4 software package. All figures representing microbial contamination were generated using the R statistical package (R Core Team, 2014).
2.11. Spatial analysis Spatial analyses were performed using drinking water distribution system design schematics available through the San Cristobal Municipality. The design schematics were often incomplete and occasionally self-contradictory, in part because of the ongoing infrastructure improvement project. Therefore, results of the drinking water analysis were aggregated by neighborhood and mapped to determine if any general spatial trends could be visually observed. The neighborhoods included in this analysis are defined based on local government records and are identified herein by their numerical identifiers: (1) Playa Mann; (2) Central; (3) Barrio Frio; (4) San Francisco; (5) Estacion Terrena; (6) Algarrobos; (7) Fragatas; (8) Albatros; (9) Cactus; (10) Penas Bajas; (11) Penas Altas; (12) Divino Nino; (13) Manatial and Isla Sur; (14) Manzanillo; (15) Palmieras; (16) El Progreso; and (17) Socavon. Neighborhoods 14 and 15 are along the eastern border of the main population center (Puerto Baquerizo Moreno), which contains neighborhoods 1 through 13 (Fig. 1). The second population center (El Progreso), which is found in the highlands, includes neighborhoods 16 and 17.
Fig. 2. Box plots of Log10 transformed total coliform concentrations aggregated by neighborhood. San Cristobal Island, Galápagos (2013–2014). Box plots show median values (solid horizontal line), with the upper and lower quartiles denoted by the hinges of the box, the maximum and minimum values denoted by vertical lines, and any outliers denoted by dots. Median values above 2 Log10 E. coli were considered very high risk. Neighborhoods tested include: (1) Playa Mann; (2) Central; (3) Barrio Frio; (4) San Francisco; (5) Estacion Terrena; (6) Algarrobos; (7) Fragatas; (8) Albatros; (9) Cactus; (10) Penas Bajas; (11) Penas Altas; (12) Divino Nino; (13) Manatial and Isla Sur; (14) Manzanillo; (15) Palmieras; (16) El Progreso; and (17) Socavon.
3. Results The fecal indicator bacteria E. coli were measured in drinking water on San Cristobal Island at the freshwater sources, immediately post-treatment, and at 50 sites in the distribution system over the course of two years. The freshwater sources of drinking water had detectable concentrations of E. coli in every sample and all samples were above the upper limit of quantification for total coliforms (>2400 MPN per 100 mL). In 2013, the E. coli geometric mean concentrations (95% CI) were: Los Americanos 269 (268–270) MPN per 100 mL (n = 2) and Cerro Gato 893 (892–894) MPN per 100 mL (n = 2). In 2014, the E. coli concentrations were: Los Americanos 82 (80–83) MPN per 100 mL (n = 4) and Cerro Gato 154 (151–156) MPN per 100 mL (n = 4). Construction of the new DWTP was completed in September 2013. As expected with the construction of a new DWTP, there was a significant decrease in microbial contamination levels at the points-of-use between 2013 and 2014 (p < 0.0001). The microbial data indicates a decrease in concentrations of total coliforms (Fig. 2) and E. coli (Fig. 3) at the neighborhood levels. These trends represent a significant decrease in concentrations of total coliforms (two orders of magnitude) and E. coli (one order of magnitude) at pointsof-use aggregated throughout the distribution system (p < 0.0001). Three neighborhoods saw a decrease in microbial contamination, yet retained some overlapping data points in the upper and lower extremes of each year (Figs. 2 and 3). These neighborhoods were not proximally located and had no clear infrastructural relationship in regards to the ongoing distribution infrastructure improvement project. The total coliform concentrations were quantifiable above the LLOD (1 MPN per 100 mL) at every point-of-use in 2013 before construction of the DWTP. After the plant began operations, the concentration of total coliforms in the finished water of the DWTP was consistently below the LLOD; however, 66% of
Fig. 3. Box plots of Log10 transformed E. coli concentrations aggregated by neighborhood. San Cristobal Island, Galápagos (2013–2014). Box plots show median values (solid horizontal line), with the upper and lower quartiles denoted by the hinges of the box, the maximum and minimum values denoted by vertical lines, and any outliers denoted by dots. Median values above 2 Log10 E. coli were considered very high risk. Neighborhoods tested include: (1) Playa Mann; (2) Central; (3) Barrio Frio; (4) San Francisco; (5) Estacion Terrena; (6) Algarrobos; (7) Fragatas; (8) Albatros; (9) Cactus; (10) Penas Bajas; (11) Penas Altas; (12) Divino Nino; (13) Manatial and Isla Sur; (14) Manzanillo; (15) Palmieras; (16) El Progreso; and (17) Socavon.
post-distribution sites (n = 50) had detectable levels of microbial contamination in at least one of the three collection cycles in 2014. The total coliform concentration measured at the points-ofuse significantly declined after the construction of the DWTP (p < 0.0001). The geometric mean of total coliform concentrations in water at the points-of-use was 1262 (1259–1265) MPN per 100 mL in June/July 2013 and 6.9 (0–18.7) MPN per 100 mL in June/July 2014 (Fig. 4). The geometric mean of E. coli concentrations in the drinking water of households was 40 (37–43) MPN per 100 mL in June/July 2013 and 1.6 (0–4.7) MPN per 100 mL in June/July 2014 (p < 0.0001) (Fig. 5). Before construction of the DWTP, E. coli concentrations were detectable during at least one collection period at every site (100%) included in this study. The post-construction percentage of sites with detectable E. coli concentrations in at least one
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Fig. 4. Spatial representation of total coliform MPN per 100 mL. San Cristobal Island, Galapagos (2013–2014).
Fig. 5. Spatial representation of Escherichia coli MPN per 100 mL. San Cristobal Island, Galapagos (2013–2014).
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W.A. Gerhard et al. / International Journal of Hygiene and Environmental Health xxx (2017) xxx–xxx Table 1 Health risks of microbial contamination before and after intervention for all tested sample sites on San Cristobal Island, Galapagos (2013–2014). Level of Riska
2013 (n = 50)
2014 (n = 50)
Low Moderate High Very High No samples
0 (0%) 4 (8%) 36 (72%) 9 (18%) 1 (2%)
36 (72%) 8 (16%) 3 (6%) 1 (2%) 0 (0%)
a Risk levels defined according to WHO Guidelines for Drinking Water Quality (WHO, 2011) as (E. coli MPN per 100 mL): Low (<1), Moderate (1–10), High (11–100), and Very High (>100).
collection period dropped to 28% of sites (n = 50). In addition, 12% of sites (n = 49) had detectable E. coli concentrations during the final cycle in 2014 when the samples for molecular analysis were collected. There was no significant relationship observed between E. coli concentrations and pH, dissolved oxygen, conductivity, or temperature. These parameters across all points-of-use were (arithmetic mean ± standard deviation): pH (7.81 ± 0.44), dissolved oxygen (6.3 ± 1.0 mg/L [78 ± 12% saturation]), conductivity (110 ± 27 mhos/cm), and temperature (26.1 ± 1.6 ◦ C). In addition, insufficient chlorine residual for disinfection was recorded at all sites along the distribution system. The log-based E. coli concentrations defined in the legends (Table 1, Fig. 5) correlate to World Health Organization (WHO) classifications of associated health risk for E. coli concentrations in drinking water (WHO, 2011). The percentage of sites with a high or very high associated health risk according to this scale dropped from 90% in 2013 to 8% in 2014 (n = 50). In addition, 80% of sites (n = 50) were reclassified to a lower level of associated risk from 2013 to 2014 (Table 1, Fig. 5). Breakdown of the sites into risk categories after the completion of the drinking water plant can be found in Table 1.
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Assessment of the HF183 assays used in this study (HF183 Endpoint, HF183 SYBR, and HF183 TaqMan) was performed on stool samples to confirm that the Bacteroides HF183 marker is present in the local population (Table 2). Among 22 human fecal samples, 18 (82%) were positive using HF183 Endpoint, 14 (64%) were positive using HF183 SYBR, and 14 (64%) were positive using HF183 TaqMan marker. Additionally, 100% (n = 6) of the tested wastewater samples were positive for the marker in all three assays. This study found that the prevalence of Bacteroides in the local population of San Cristobal Island was comparable to the prevalence reported in the literature for geographical regions where the assays were developed (Seurinck et al., 2005). None of the three HF183 assays detected human-specific Bacteroides in the finished water of the DWTP. Neither freshwater source had detectable human-specific Bacteroides despite high concentrations of E. coli. Although there was E. coli contamination measured in post-distribution samples, the HF183 SYBR assay did not detect human-specific contamination at any points-of-use. The HF183 TaqMan assay detected human-specific contamination in one point-of-use sample (5.21 ± 0.94 × 106 copies per L). The HF183 Endpoint assay initially found that five of the post-distribution sites (n = 49) were positive for human-specific Bacteroides; however, a repeated HF183 Endpoint analysis using the same extracts found reproducible positive results in only two of the five sites. This inconsistency was likely caused by target sequence concentrations in the samples near the LLOD. Of the two consistently positive samples measured via HF183 Endpoint, only one had detectable levels of E. coli contamination on any of the three sample collection periods in 2014. The PCR products from the two consistently positive samples were then examined using Sanger DNA sequencing. Only the site with detectable E. coli contamination was confirmed to have a sequence belonging to the genus Bacteroides based on Sanger sequencing results and a BLAST search of the NCBI database. The other sequence was unknown. The site with confirmed Bacteroides was the same site with positive TaqMan assay
Table 2 Human-specific HF183 Bacteroides assay results for human fecal samples of residents and raw sewage samples on San Cristobal Island, Galapagos (2014). Sample
HF183 Endpoint Conventional PCR
HF183 SYBR human-specific Bacteroides markers per gram of wet feces or per liter of influent (mean ± SD)
HF183 TaqMan human-specific Bacteroides markers per gram of wet feces or per liter of influent (mean ± SD)
Human 1 Human 2 Human 3 Human 4 Human 5 Human 6 Human 7 Human 8 Human 9 Human 10 Human 11 Human 12 Human 13 Human 14 Human 15 Human 16 Human 17 Human 18 Human 19 Human 20 Human 21 Human 22 Sewage 1 (6/24) Sewage 2 (6/24) Sewage 3 (6/24) Sewage 4 (7/18) Sewage 5 (7/18) Sewage 6 (7/18)
ND + + + + + ND + + + + + + + ND ND + + + + + + + + + + + +
ND ND 1.44 ± 1.44 × 105 3.88 ± 0.39 × 107 ND 3.50 ± 0.84 × 106 ND 1.12 ± 0.58 × 1010 2.63 ± 0.73 × 107 ND ND 5.60 ± 3.8 × 106 2.93 ± 2.93 × 104 6.00 ± 4.85 × 104 5.70 ± 0.23 × 104 2.75 ± 1.14 × 102 ND 5.83 ± 0.26 × 105 3.78 ± 4.30 × 104 4.68 ± 0.75 × 108 ND 2.20 ± 0.50 × 106 2.94 ± 0.16 × 106 3.00 ± 0.19 × 107 7.94 ± 0.59 × 106 5.06 ± 0.69 × 108 3.00 ± 0.34 × 108 3.62 ± 0.10 × 108
ND ND 2.14 ± 0.17 × 105 4.73 ± 0.37 × 106 1.02 ± 0.14 × 108 2.03 ± 0.06 × 106 ND 1.84 ± 0.06 × 109 2.35 ± 0.27 × 106 ND 8.33 × 103 1.84 ± 0.24 × 106 ND 1.72 ± 0.02 × 107 1.03 ± 0.18 × 104 1.31 ± 0.34 × 104 7.33 ± 6.48 × 103 ND ND 9.88 ± 0.45 × 107 ND 4.63 ± 0.28 × 105 2.65 ± 0.14 × 109 1.71 ± 0.04 × 1010 6.49 ± 0.64 × 109 1.88 ± 0.07 × 1011 1.40 ± 0.06 × 1011 1.67 ± 0.06 × 1011
ND, Not Detected.
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results, and was the only site counted as positive for human source contamination.
4. Discussion The study provided an opportunity to evaluate water quality before and after construction of a new DWTP in an area facing developmental pressure. A significant decrease in microbial contamination was observed across the distribution system after the replacement of the DWTP with a concomitant decrease in associated health risks as defined by the WHO. The finished water at the DWTP was consistently free of indicator bacteria. However, improvement to finished water alone was not sufficient to prevent contamination at points of use. This study still found contamination during at least one 2014 collection cycle in 66% of point-of-use sites as measured by total coliforms and in 28% of sites as measured by E. coli (n = 50). Despite the construction of a DWTP, problems with the distribution infrastructure or with post-distribution storage at the household level allow for contamination or possibly regrowth of fecal indicator bacteria. This study provides further evidence that a holistic approach to drinking water infrastructure improvement is necessary to consistently achieve concentrations of indicator bacteria deemed safe by the WHO. The observation that drinking water quality can deteriorate between treatment and the point-of-use is consistent with studies in other parts of South America (Levy et al., 2008; Rufener et al., 2010), Asia (Eshcol et al., 2009; Kær Jensen et al., 2002), and Africa (Clasen and Bastable, 2003). In many of these studies, drinking water handling and storage at the household level was highlighted as a potential area for improvement (Noble et al., 2003). This study also explored potential sources of drinking water contamination and confirmed human carriage of the Bacteroides HF183 MST marker in the Galapagos Islands. Because the Galapagos are so geographically remote, the prevalence of these markers in the intestinal flora of archipelago residents lends credence to the idea that they will be found in various remote populations around the globe. Although validation studies should always be performed prior to use of MST assays in a new population, these findings may have implications to other geographically distinct populations affected by poor drinking water quality such as those found in many developing world countries. To our knowledge, this study is the first to use MST markers in South America to determine specific sources of fecal indicator bacteria in drinking water. The combination of conventional and alternative methods found in this study generates a more complete picture of drinking water contamination than is typically found in the literature and provides useful data for planning and remediation. Although fecal indicator bacteria were found in drinking water at the point-of-use, the lack of human-specific MST markers raises questions about the origin of observed contamination and the remediation steps that are most likely to be effective. It is not clear whether the observed E. coli contamination is truly fecal in origin or perhaps the result of environmental regrowth as has been reported previously for soil and surface waters in other tropical/subtropical regions (Fujioka and Unutoa, 2006; Toranzos, 1991). The detection of FIB in the absence of human-source markers could also suggest that fecal contamination from non-human animals is able to infiltrate the system, which would also be a health concern. Expanding the conventional and alternative analysis of drinking water to include pre- and post-storage at the household level could provide further insight to possible sources of contamination and the most appropriate steps to achieve drinking water free of E. coli. This study would have been improved by including measurements throughout the year to account for seasonal variation including changes in precipitation. Because of personnel and
funding limitations, the study could only be performed during the months of June and July in consecutive years. This time period occurs during the initial months of the dry season, which results in less rainwater runoff that may contribute to bacterial transport through the environment. Future research could strengthen the findings of this report by examining drinking water quality differences between the wet (December to May) and dry (June to November) seasons. In conclusion, this study demonstrates that drinking water handling and storage are critical in maintaining microbial water quality at points of use. Both treatment and distribution should be considered when planning water infrastructure improvements in developing and developed countries. Contamination or regrowth post-treatment likely explains much of the observed variation between sites. Improving the rate of compliance to the cleaning and maintenance schedules recommended by the municipality may improve drinking water quality. Education programs to teach the public about the importance of proper care and maintenance for household water cisterns may be useful. In the long term, a plan to construct neighborhood-level cisterns will move the maintenance and cleaning tasks from local residents to municipal employees. The shift of responsibility for daily compliance tasks may improve post-treatment drinking water handling and storage practices island-wide, thereby improving drinking water quality at the points-of-use. Finally, 24 h operation of the drinking water distribution system at pressure could create more predictable residence times, reduce the likelihood of contamination during distribution, and improve the ability to accurately dose chlorine to the system. Available water quantity may preclude this approach during certain times of the year; however, seasonal implementation may be feasible. Conflict of interest declaration The authors declare they have no actual or potential conflicts of interest. Acknowledgments The authors would like to acknowledge the support of several programs within the University of North Carolina for their support: the Jon Curtis Student Enrichment Fund - Summer Scholarship; the Center for Global Initiatives - Vimy Global Team Award; and the Carolina Center for Public Service - Hyatt Rotary Public Service Award and Sherpa Fellowship. In addition, the logistical and financial assistance provided through the Galapagos Science Center and the UNC Center for Galapagos Studies was invaluable to the completion of this project. Finally, the authors would like to thank Shannon Steel for her contributions to visualizing these data through GIS. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijheh.2017.01. 010. References ¨ Åstrom, J., Pettersson, T.J.R., Reischer, G.H., Norberg, T., Hermansson, M., 2015. Incorporating expert judgments in utility evaluation of bacteroidales qpcr assays for microbial source tracking in a drinking water source. Environ. Sci. Technol. 49, 1311–1318, http://dx.doi.org/10.1021/es504579j. Bain, R., Cronk, R., Wright, J., Yang, H., Slaymaker, T., Bartram, J., 2014. Fecal contamination of drinking-water in low- and middle-income countries: a systematic review and meta-analysis. PLoS Med. 11, http://dx.doi.org/10.1371/ journal.pmed.1001644.
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Water quality at points-of-use in the Galapagos Islands William A. Gerhard a,∗ , Wan Suk Choi b , Kelly M Houck c , Jill R Stewart a a Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina, 170 Rosenau Hall, CB #7400, 135 Dauer Drive, 27599-7400 Chapel Hill, NC, USA b Department of Biostatistics, Gillings School of Global Public Health, University of North Carolina, 170 Rosenau Hall, CB #7400, 135 Dauer Drive, 27599-7400 Chapel Hill, NC, USA c Department of Anthropology, University of North Carolina, 301 Alumni Building, CB #3115, UNC-CH, 27599-3115 Chapel Hill, NC, USA
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Article history: Received 1 September 2016 Received in revised form 14 December 2016 Accepted 26 January 2017 Keywords: Escherichia coli Bacteroides Fecal coliforms Regrowth Post distribution contamination Microbial source tracking
a b s t r a c t Piped drinking water is often considered a gold standard for protecting public health but research is needed to explicitly evaluate the effect of centralized treatment systems on water quality in developing world settings. This study examined the effect of a new drinking water treatment plant (DWTP) on microbial drinking water quality at the point-of-use on San Cristobal Island, Galapagos using fecal indicator bacteria total coliforms and Escherichia coli. Samples were collected during six collection periods before and after operation of the DWTP began from the freshwater sources (n = 4), the finished water (n = 6), and 50 sites throughout the distribution system (n = 287). This study found that there was a significant decrease in contamination by total coliforms (two orders of magnitude) and E. coli (one order of magnitude) after DWTP operation began (p < 0.001). However, during at least one post-construction collection cycle, total coliforms and E. coli were still found at 66% and 28% of points-of-use (n = 50), respectively. During the final collection period, conventional methods were augmented with human-specific Bacteroides assays – validated herein – with the goal of elucidating possible microbial contamination sources. Results show that E. coli contamination was not predictive of contamination by human wastes and suggests that observed indicator bacteria contamination may have environmental origins. Together these findings highlight the necessity of a holistic approach to drinking water infrastructure improvements in order to deliver high quality water through to the point-of-use. © 2017 Elsevier GmbH. All rights reserved.
1. Introduction Diarrheal diseases are the sixth-leading cause of premature death worldwide, above tuberculosis, malaria, and traffic accidents (Lopez et al., 2006). Diarrhea can be caused by a range of viral, bacterial, and protozoan pathogens, which increases the complexity of addressing this problem (Fischer-Walker et al., 2013). The United Nations aimed to halve the proportion of the world population without sustainable access to drinking water between the years of 1990 and 2015 (United Nations, 2015). As a result, nearly two billion people gained access to piped drinking water (United
Abbreviations: DWTP, drinking water treatment plant; FIB, fecal indicator bacteria; LLOD, lower limit of detection; MPN, most probable number; MST, microbial source tracking; WHO, World Health Organization. ∗ Correspondence to: 170 Rosenau Hall, CB #7400, 135 Dauer Drive, 27599-7400 Chapel Hill, NC, USA. E-mail addresses: [email protected] (W.A. Gerhard), [email protected] (W.S. Choi), [email protected] (K.M. Houck), [email protected] (J.R. Stewart).
Nations, 2015). These improvements will no doubt improve public health. However, research is needed to explicitly evaluate the effect of piped drinking water on point-of-use water quality in developing world settings. It is possible for drinking water quality to deteriorate between treatment and the point-of-use, in which case the false presumption of safety may inadvertently increase the public health risk of water-borne disease (Bain et al., 2014; Rufener et al., 2010; Wright et al., 2004). For example, it has been reported that access to protected sources may reduce the rate at which consumers observe precautions such as boiling drinking water prior to consumption (Lindskog and Lindskog, 1988). As a result, improving access to drinking water without ensuring its quality may have unintended negative impacts on human health. This study examined point-of-use water quality on the Galapagos Islands of Ecuador, an area with piped drinking water that is also undergoing infrastructure improvements. Ecotourism to the islands has grown exponentially from 2000 tourists in the year 1960 to 120,000 tourists in 2005 (Epler, 2007). The popularity of ecotourism creates a framework called the Galapagos Paradox where increasing strain is placed on the “pristine” environment that many
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tourists hope to enjoy (Quiroga, 2009). Among other stressors, the strain that tourism places on the drinking water infrastructure has contributed to drinking water quality problems that have persisted for decades (Liu and D’Ozouville, 2013; Reyes et al., 2015). Effective drinking water infrastructure requires access to quality source water. On San Cristobal Island, the source water is from two freshwater lakes – La Toma de Los Americanos (Los Americanos) and Cerro Gato – in the highlands of the island. These two sources are fed by rainwater during the rainy season and have sufficient capacity to provide 30 L per second of water flow to the drinking water treatment plant (DWTP) year-round through the dry season. San Cristobal draws its drinking water entirely from these surface reservoirs rather than subsurface reservoirs like two of the other inhabited islands on the archipelago – Santa Cruz and Isabela. A project to replace the DWTP and distribution system on San Cristobal Island is underway. Construction of a new DWTP began in late 2012, and the new DWTP began operations in September 2013. This DWTP includes treatment processes common to modern drinking water treatment systems including rapid mixing, coagulation, flocculation, sedimentation, filtration, and chlorine gas disinfection. An infrastructure improvement project is ongoing as the drinking water distribution infrastructure is replaced; however, several problems that may allow for post-collection contamination continue to affect the distribution system. The distribution system is not consistently pressurized, instead operating as a gravity-fed system a few hours each day. Negative hydraulic pressure associated with interrupted service can draw contamination into water pipes from the surrounding environment (Lee and Schwab, 2005). Municipal sewer lines have also been laid immediately below the drinking water distribution system in the ongoing infrastructure improvement project. The vast majority (79%) of households are connected to municipal sewer lines for waste management while a smaller proportion (19%) of households uses septic tanks. The wastewater treatment plant on the island was not in operation during the time period of this study, and wastewater from the piped system was discharged directly to the ocean. For houses with septic tanks, the volcanic rock conditions likely prevent septic fields from functioning properly and may allow inadvertent contamination resulting from negative hydraulic pressure associated with inconsistent water delivery. Lack of consistent water availability necessitates the use of storage cisterns at points-of-use. Several studies have found increases in contamination during household storage and at least one study has identified the household level as the point at which the largest impact on public health can be made (Clasen and Bastable, 2003; Eshcol et al., 2009; Kær Jensen et al., 2002; Levy et al., 2008; Rufener et al., 2010; Wright et al., 2004). The cisterns on San Cristobal were above-ground plastic storage tanks. These cisterns are filled from the top by hoses that shutoff using a simple fill valve, so the opportunity for feedback into the distribution from cisterns is eliminated. Domestic animals are often near the cisterns on household property and may represent a possible route of contamination, but the likelihood of this contamination is reduced given the overwhelming majority of cisterns were covered. Variable cistern residence times also complicate the calculation of doses for residual disinfection of finished water. Despite the improvements made to the infrastructure, lack of consideration for the effects of intermittent water delivery and point-of-use water storage may allow bacteria in the environment to infiltrate the system and, under certain conditions, multiply. This study establishes baseline measurements of drinking water quality at various points of the distribution system on the island of San Cristobal over a two year period and provides the most comprehensive spatial representation of microbial drinking water quality on any island in the Galapagos archipelago to
date. Furthermore, this study examines the effect of a newly constructed drinking water treatment plant on microbial water quality, both in terms of comparisons between years and in terms of compliance with established World Health Organization (WHO) Guidelines. This study also evaluates the sources of observed fecal contamination through microbial source tracking (MST) methods. Because of spatially widespread microbial contamination observed during the first year of the study, human-specific assays were used to narrow the list of possible sources for microbial contamination. Several studies have applied this approach to drinking water source quality monitoring; however, few have applied it to drinking water qual¨ et al., 2015; ity monitoring through the distribution system (Åstrom Marti et al., 2013). This is one of the first studies to apply MST methods to examine drinking water quality at the point-of-use in South America.
2. Methods 2.1. Site selection The study area of San Cristobal Island includes the two main population centers: El Progreso (population of 500, altitude 700 m) and Puerto Baquerizo Moreno (population of 5400, altitude 0–70 m) (Fig. 1). This study also included the freshwater sources (Los Americanos and Cerro Gato) and the DWTP (altitude 700 m) – all located on San Cristobal. During June 2013, 50 sample sites from 15 neighborhoods on the island of San Cristobal were selected to represent locations throughout the drinking water distribution system. Each of these sites was also subject to post-distribution storage – typically in the form of rooftop or ground-level cisterns where water was stored prior to household use.
2.2. Sample collection A total of 143 point-of-use water samples from 50 sites along the distribution system were collected in three collection periods, which occurred in 7–10 day intervals in June and July 2013. A total of 144 samples from the same 50 sites were collected during three collection periods at the same intervals during the same months in 2014. Every effort was made to collect a sample from each site during each collection period, but inconsistent business hours and shifting schedules occasionally prevented collection from sites within 48 h of the start of a collection period. In these instances, the site was skipped for that collection period. The two freshwater sources for the island (Los Americanos and Cerro Gato) were included in the study to assess microbial contamination from the beginning to the end of the treatment and distribution processes. Construction of the new DWTP using a treatment process involving flocculation, sedimentation, rapid filtration, and chlorine-gas disinfection was completed in September 2013. Samples were collected weekly from the new DWTP in 2014. Water samples of 100 mL were collected in vacuum sealed, sterile 120 mL vessels containing sodium thiosulfate from all 50 sample collection sites during each collection period. The samples were placed in a cooler with ice packs and taken to the Galapagos Science Center for enumeration of total coliform and E. coli using ® the IDEXX Colilert method (Westbrook, ME USA) within 12 h of sample collection. Permit requirements limited the number of collection opportunities at the freshwater sources; however, there was one collection period in 2013 and three collection periods in 2014. Water (500 mL) was collected from each point-of-use during the final collection period in 2014 in double-washed and autoclaved glass laboratory bottles for MST analysis.
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Fig. 1. Inhabited zones and neighborhoods of San Cristobal Island and neighborhoods of Puerto Baquerizo Moreno.
2.3. Total coliform and E. coli enumeration
2.4. Physical and chemical parameters
Total coliform and E. coli most probable number (MPN) per 100 mL were measured at the Galapagos Science Center ® using IDEXX Colilert (Westbrook, ME USA) according to the manufacturer’s protocol (Microbial Contaminants Method 9223, 2005).
All physical and chemical parameters were measured in 2014 using a YSI Professional Plus handheld multi-parameter instrument. Atmospheric pressure was measured using the internal barometer and temperature, dissolved oxygen, conductivity, and pH were all measured using the relevant YSI probe attachments.
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Recalibration of all probes was performed using external and internal standards before each collection period. Data were not collected in 2013 because appropriate instrumentation was not available on the island at that time. Chlorine residuals and water temperature in the distribution pipeline were not measured because direct access to the pipeline was not possible. In addition, there was not capacity to perform chlorine measurements in either year of the study. 2.5. DNA extraction and storage The 500 mL samples collected for MST analysis were filtered through 0.45 m polycarbonate filter paper using a vacuum apparatus. The resulting filter paper was stored in a −20 ◦ C freezer prior to transport to the University of North Carolina at Chapel Hill (UNC-CH) for DNA extraction and analysis. DNA extraction with performed in September 2014, two months after collection. DNA extraction was performed on all filtered samples using the ® MoBio PowerSoil DNA Isolation Kit (Carlsbad, CA USA) accord® ing to the manufacturer’s protocol. PowerSoil was chosen instead ® of PowerWater because prior studies show that the former can effectively prevent inhibition (Cox and Goodwin, 2013). In addition, studies have shown similar recovery with the two MoBio kits when using filter homogenized samples (Kaevska and Slana, 2015). The 100 L DNA extract was transferred into two single-use aliquots of 20 L and one reserve aliquot of 60 L which were stored at −80 ◦ C until they were examined with the PCR/qPCR assays beginning in February 2015, seven months after collection. 2.6. Conventional PCR Conventional PCR was performed at UNC-CH using the humanspecific HF183 Endpoint PCR assay as described by Bernhard and Field with slight modifications (Bernhard and Field, 2000). These modifications deviated from the initial study protocol primarily in the development of the master mix. The HF183 Endpoint assay was performed using the following master mix components (stock concentrations): HF183F (100 M), Bac708R (100 M), and 5 PRIME MasterMix (2.5×). The optimized final concentration of these reagents and the amount of reagent per reaction are found in Table S1. Forward and reverse primer sequences for this assay are described in Table S4.
at 60 ◦ C for 60 s, and a plate read; and (4) A melt curve of 60 ◦ C to 94.8 ◦ C, with steps of 0.4 ◦ C every 10 s. The HF183 TaqMan assay was performed using the following master mix components (stock concentrations): HF183F (100 M), BthetR1 (100 M), BthetP1 (100 M), and Fast Universal Master Mix (2×). The master mix was made in two steps: (1) Creating a primer/probe mixture from the stock primers/probes; and (2) Combining this prepared primer/probe mixture with the remaining master mix reagents. The optimized amount of reagent per reaction well is found below (Table S3). 2.8. HF183 prevalence Stool samples were collected from 22 residents of San Cristobal Island as part of an ongoing biological anthropology study. Informed consent was obtained, and the study protocol and analysis were approved by the Institutional Review Boards at the University of North Carolina at Chapel Hill and the Universidad San Francisco ® de Quito. Using the Omega Bio-Tek E.Z.N.A. Stool DNA Kit (Norcross, GA, USA), DNA was extracted from 200 mg of wet stool and eluted to a final volume of 50 L. These samples were analyzed using the conventional PCR and qPCR methods outlined above to determine the prevalence of human-specific Bacteroides in the local population. Six 100 mL samples were collected from the influent to the waste water treatment plant (WWTP) on two different dates (24 June 2014 and 18 July 2014) to examine the concentration of human-specific Bacteroides in raw sewage. Of these samples, 10 mL from each waste water sample was filtered and analyzed using the same DNA extraction protocol and PCR/qPCR assays as the drinking water samples. 2.9. DNA sequencing Positive point-of-use samples were confirmed via Sanger sequencing performed by Eton Biosciences Incorporated (Research Triangle Park, NC). The resulting DNA sequences were compared to known microbial genomes using the Basic Local Alignment Search Tool (BLASTN 2.2.31) available online through the National Center for Biotechnical Information (NCBI) (Zhang et al., 2000). The sequences were searched in the 16S ribosomal RNA sequences (Bacteria and Archaea) genome database.
2.7. Quantitative real-time PCR 2.10. Data analysis All quantitative real-time PCR (qPCR) was performed at UNC-CH using two human-specific HF183 assays (HF183 SYBR and HF183 TaqMan) (Haugland et al., 2010; Seurinck et al., 2005). Both assays were performed in a Bio-Rad CFX96 Touch Real-Time PCR Detection System (Hercules, CA, USA), which includes the Bio-Rad CFX96 Optical Reaction Module and the Bio-Rad C1000 Touch Thermal Cycler. The standard curve was calculated using DNA from Bacteroides dorei (GenBank reference AB242142) (Bernhard and Field, 2000). This DNA was serially diluted with copy number concentrations ranging from 108 per L to 101 per L. An eight-point standard curve using these concentrations in triplicate was generated for each run. The HF183 SYBR assay was performed using the following master mix components (stock concentrations): HF183F (100 M), HFsybR (100 M), dNTP with dUTP (5 mM), MgCl2 (50 mM), PCR 10× reaction buffer (10×), and Hot GoldStar DNA Polymerase (5 U/L). The optimized final concentration of these reagents and the amount of reagent per reaction well is found below (Table S2). The cycling conditions for HF183 SYBR were as follows: (1) 50 ◦ C for 2 min; (2) Polymerase activation at 95 ◦ C for 10 min; (3) 40 cycles of denaturation at 95 ◦ C for 30 s, annealing at 53 ◦ C for 60 s, extension
All microbial data (total coliforms and E. coli) was logarithmically transformed for data analysis. Microbial data below the lower limit of detection (1 MPN per 100 mL) were assigned the √ ) where LLOD represents the lower limit of detection. value ( LLOD 2
This method is widely used to generate fill-in values for samples below the LLOD when data are not highly skewed (Finkelstein and Verma, 2001). This method is appropriate for this study because the data have been log-transformed to better approximate normal distribution. Samples above the upper limit of quantification were assigned the value of this upper limit (2419.6 MPN per 100 mL). The arithmetic mean of the logarithmically transformed data was calculated for each site for 2013 and 2014. These values were compared between the years using a Wilcoxon Ranked Sum Test to establish the significance of differences. The hypotheses tested included: (1) H0 : T2013 = T2014 , where T2013 and T2014 imply arithmetic mean of ln(total coliforms) of all sites in 2013 and 2014, respectively; and (2) H0 : E2013 = E2014 , where E2013 and E2014 imply arithmetic mean of ln (E. coli) of all sites in 2013 and 2014, respectively. The hypothesis testing was performed using a 0.05 significance level (␣ = 0.05).
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A regression analysis was performed to determine if there was any relationship between microbial contaminant concentration and pH or water temperature. These analyses were performed with the SAS 9.4 software package. All figures representing microbial contamination were generated using the R statistical package (R Core Team, 2014).
2.11. Spatial analysis Spatial analyses were performed using drinking water distribution system design schematics available through the San Cristobal Municipality. The design schematics were often incomplete and occasionally self-contradictory, in part because of the ongoing infrastructure improvement project. Therefore, results of the drinking water analysis were aggregated by neighborhood and mapped to determine if any general spatial trends could be visually observed. The neighborhoods included in this analysis are defined based on local government records and are identified herein by their numerical identifiers: (1) Playa Mann; (2) Central; (3) Barrio Frio; (4) San Francisco; (5) Estacion Terrena; (6) Algarrobos; (7) Fragatas; (8) Albatros; (9) Cactus; (10) Penas Bajas; (11) Penas Altas; (12) Divino Nino; (13) Manatial and Isla Sur; (14) Manzanillo; (15) Palmieras; (16) El Progreso; and (17) Socavon. Neighborhoods 14 and 15 are along the eastern border of the main population center (Puerto Baquerizo Moreno), which contains neighborhoods 1 through 13 (Fig. 1). The second population center (El Progreso), which is found in the highlands, includes neighborhoods 16 and 17.
Fig. 2. Box plots of Log10 transformed total coliform concentrations aggregated by neighborhood. San Cristobal Island, Galápagos (2013–2014). Box plots show median values (solid horizontal line), with the upper and lower quartiles denoted by the hinges of the box, the maximum and minimum values denoted by vertical lines, and any outliers denoted by dots. Median values above 2 Log10 E. coli were considered very high risk. Neighborhoods tested include: (1) Playa Mann; (2) Central; (3) Barrio Frio; (4) San Francisco; (5) Estacion Terrena; (6) Algarrobos; (7) Fragatas; (8) Albatros; (9) Cactus; (10) Penas Bajas; (11) Penas Altas; (12) Divino Nino; (13) Manatial and Isla Sur; (14) Manzanillo; (15) Palmieras; (16) El Progreso; and (17) Socavon.
3. Results The fecal indicator bacteria E. coli were measured in drinking water on San Cristobal Island at the freshwater sources, immediately post-treatment, and at 50 sites in the distribution system over the course of two years. The freshwater sources of drinking water had detectable concentrations of E. coli in every sample and all samples were above the upper limit of quantification for total coliforms (>2400 MPN per 100 mL). In 2013, the E. coli geometric mean concentrations (95% CI) were: Los Americanos 269 (268–270) MPN per 100 mL (n = 2) and Cerro Gato 893 (892–894) MPN per 100 mL (n = 2). In 2014, the E. coli concentrations were: Los Americanos 82 (80–83) MPN per 100 mL (n = 4) and Cerro Gato 154 (151–156) MPN per 100 mL (n = 4). Construction of the new DWTP was completed in September 2013. As expected with the construction of a new DWTP, there was a significant decrease in microbial contamination levels at the points-of-use between 2013 and 2014 (p < 0.0001). The microbial data indicates a decrease in concentrations of total coliforms (Fig. 2) and E. coli (Fig. 3) at the neighborhood levels. These trends represent a significant decrease in concentrations of total coliforms (two orders of magnitude) and E. coli (one order of magnitude) at pointsof-use aggregated throughout the distribution system (p < 0.0001). Three neighborhoods saw a decrease in microbial contamination, yet retained some overlapping data points in the upper and lower extremes of each year (Figs. 2 and 3). These neighborhoods were not proximally located and had no clear infrastructural relationship in regards to the ongoing distribution infrastructure improvement project. The total coliform concentrations were quantifiable above the LLOD (1 MPN per 100 mL) at every point-of-use in 2013 before construction of the DWTP. After the plant began operations, the concentration of total coliforms in the finished water of the DWTP was consistently below the LLOD; however, 66% of
Fig. 3. Box plots of Log10 transformed E. coli concentrations aggregated by neighborhood. San Cristobal Island, Galápagos (2013–2014). Box plots show median values (solid horizontal line), with the upper and lower quartiles denoted by the hinges of the box, the maximum and minimum values denoted by vertical lines, and any outliers denoted by dots. Median values above 2 Log10 E. coli were considered very high risk. Neighborhoods tested include: (1) Playa Mann; (2) Central; (3) Barrio Frio; (4) San Francisco; (5) Estacion Terrena; (6) Algarrobos; (7) Fragatas; (8) Albatros; (9) Cactus; (10) Penas Bajas; (11) Penas Altas; (12) Divino Nino; (13) Manatial and Isla Sur; (14) Manzanillo; (15) Palmieras; (16) El Progreso; and (17) Socavon.
post-distribution sites (n = 50) had detectable levels of microbial contamination in at least one of the three collection cycles in 2014. The total coliform concentration measured at the points-ofuse significantly declined after the construction of the DWTP (p < 0.0001). The geometric mean of total coliform concentrations in water at the points-of-use was 1262 (1259–1265) MPN per 100 mL in June/July 2013 and 6.9 (0–18.7) MPN per 100 mL in June/July 2014 (Fig. 4). The geometric mean of E. coli concentrations in the drinking water of households was 40 (37–43) MPN per 100 mL in June/July 2013 and 1.6 (0–4.7) MPN per 100 mL in June/July 2014 (p < 0.0001) (Fig. 5). Before construction of the DWTP, E. coli concentrations were detectable during at least one collection period at every site (100%) included in this study. The post-construction percentage of sites with detectable E. coli concentrations in at least one
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Fig. 4. Spatial representation of total coliform MPN per 100 mL. San Cristobal Island, Galapagos (2013–2014).
Fig. 5. Spatial representation of Escherichia coli MPN per 100 mL. San Cristobal Island, Galapagos (2013–2014).
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W.A. Gerhard et al. / International Journal of Hygiene and Environmental Health xxx (2017) xxx–xxx Table 1 Health risks of microbial contamination before and after intervention for all tested sample sites on San Cristobal Island, Galapagos (2013–2014). Level of Riska
2013 (n = 50)
2014 (n = 50)
Low Moderate High Very High No samples
0 (0%) 4 (8%) 36 (72%) 9 (18%) 1 (2%)
36 (72%) 8 (16%) 3 (6%) 1 (2%) 0 (0%)
a Risk levels defined according to WHO Guidelines for Drinking Water Quality (WHO, 2011) as (E. coli MPN per 100 mL): Low (<1), Moderate (1–10), High (11–100), and Very High (>100).
collection period dropped to 28% of sites (n = 50). In addition, 12% of sites (n = 49) had detectable E. coli concentrations during the final cycle in 2014 when the samples for molecular analysis were collected. There was no significant relationship observed between E. coli concentrations and pH, dissolved oxygen, conductivity, or temperature. These parameters across all points-of-use were (arithmetic mean ± standard deviation): pH (7.81 ± 0.44), dissolved oxygen (6.3 ± 1.0 mg/L [78 ± 12% saturation]), conductivity (110 ± 27 mhos/cm), and temperature (26.1 ± 1.6 ◦ C). In addition, insufficient chlorine residual for disinfection was recorded at all sites along the distribution system. The log-based E. coli concentrations defined in the legends (Table 1, Fig. 5) correlate to World Health Organization (WHO) classifications of associated health risk for E. coli concentrations in drinking water (WHO, 2011). The percentage of sites with a high or very high associated health risk according to this scale dropped from 90% in 2013 to 8% in 2014 (n = 50). In addition, 80% of sites (n = 50) were reclassified to a lower level of associated risk from 2013 to 2014 (Table 1, Fig. 5). Breakdown of the sites into risk categories after the completion of the drinking water plant can be found in Table 1.
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Assessment of the HF183 assays used in this study (HF183 Endpoint, HF183 SYBR, and HF183 TaqMan) was performed on stool samples to confirm that the Bacteroides HF183 marker is present in the local population (Table 2). Among 22 human fecal samples, 18 (82%) were positive using HF183 Endpoint, 14 (64%) were positive using HF183 SYBR, and 14 (64%) were positive using HF183 TaqMan marker. Additionally, 100% (n = 6) of the tested wastewater samples were positive for the marker in all three assays. This study found that the prevalence of Bacteroides in the local population of San Cristobal Island was comparable to the prevalence reported in the literature for geographical regions where the assays were developed (Seurinck et al., 2005). None of the three HF183 assays detected human-specific Bacteroides in the finished water of the DWTP. Neither freshwater source had detectable human-specific Bacteroides despite high concentrations of E. coli. Although there was E. coli contamination measured in post-distribution samples, the HF183 SYBR assay did not detect human-specific contamination at any points-of-use. The HF183 TaqMan assay detected human-specific contamination in one point-of-use sample (5.21 ± 0.94 × 106 copies per L). The HF183 Endpoint assay initially found that five of the post-distribution sites (n = 49) were positive for human-specific Bacteroides; however, a repeated HF183 Endpoint analysis using the same extracts found reproducible positive results in only two of the five sites. This inconsistency was likely caused by target sequence concentrations in the samples near the LLOD. Of the two consistently positive samples measured via HF183 Endpoint, only one had detectable levels of E. coli contamination on any of the three sample collection periods in 2014. The PCR products from the two consistently positive samples were then examined using Sanger DNA sequencing. Only the site with detectable E. coli contamination was confirmed to have a sequence belonging to the genus Bacteroides based on Sanger sequencing results and a BLAST search of the NCBI database. The other sequence was unknown. The site with confirmed Bacteroides was the same site with positive TaqMan assay
Table 2 Human-specific HF183 Bacteroides assay results for human fecal samples of residents and raw sewage samples on San Cristobal Island, Galapagos (2014). Sample
HF183 Endpoint Conventional PCR
HF183 SYBR human-specific Bacteroides markers per gram of wet feces or per liter of influent (mean ± SD)
HF183 TaqMan human-specific Bacteroides markers per gram of wet feces or per liter of influent (mean ± SD)
Human 1 Human 2 Human 3 Human 4 Human 5 Human 6 Human 7 Human 8 Human 9 Human 10 Human 11 Human 12 Human 13 Human 14 Human 15 Human 16 Human 17 Human 18 Human 19 Human 20 Human 21 Human 22 Sewage 1 (6/24) Sewage 2 (6/24) Sewage 3 (6/24) Sewage 4 (7/18) Sewage 5 (7/18) Sewage 6 (7/18)
ND + + + + + ND + + + + + + + ND ND + + + + + + + + + + + +
ND ND 1.44 ± 1.44 × 105 3.88 ± 0.39 × 107 ND 3.50 ± 0.84 × 106 ND 1.12 ± 0.58 × 1010 2.63 ± 0.73 × 107 ND ND 5.60 ± 3.8 × 106 2.93 ± 2.93 × 104 6.00 ± 4.85 × 104 5.70 ± 0.23 × 104 2.75 ± 1.14 × 102 ND 5.83 ± 0.26 × 105 3.78 ± 4.30 × 104 4.68 ± 0.75 × 108 ND 2.20 ± 0.50 × 106 2.94 ± 0.16 × 106 3.00 ± 0.19 × 107 7.94 ± 0.59 × 106 5.06 ± 0.69 × 108 3.00 ± 0.34 × 108 3.62 ± 0.10 × 108
ND ND 2.14 ± 0.17 × 105 4.73 ± 0.37 × 106 1.02 ± 0.14 × 108 2.03 ± 0.06 × 106 ND 1.84 ± 0.06 × 109 2.35 ± 0.27 × 106 ND 8.33 × 103 1.84 ± 0.24 × 106 ND 1.72 ± 0.02 × 107 1.03 ± 0.18 × 104 1.31 ± 0.34 × 104 7.33 ± 6.48 × 103 ND ND 9.88 ± 0.45 × 107 ND 4.63 ± 0.28 × 105 2.65 ± 0.14 × 109 1.71 ± 0.04 × 1010 6.49 ± 0.64 × 109 1.88 ± 0.07 × 1011 1.40 ± 0.06 × 1011 1.67 ± 0.06 × 1011
ND, Not Detected.
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results, and was the only site counted as positive for human source contamination.
4. Discussion The study provided an opportunity to evaluate water quality before and after construction of a new DWTP in an area facing developmental pressure. A significant decrease in microbial contamination was observed across the distribution system after the replacement of the DWTP with a concomitant decrease in associated health risks as defined by the WHO. The finished water at the DWTP was consistently free of indicator bacteria. However, improvement to finished water alone was not sufficient to prevent contamination at points of use. This study still found contamination during at least one 2014 collection cycle in 66% of point-of-use sites as measured by total coliforms and in 28% of sites as measured by E. coli (n = 50). Despite the construction of a DWTP, problems with the distribution infrastructure or with post-distribution storage at the household level allow for contamination or possibly regrowth of fecal indicator bacteria. This study provides further evidence that a holistic approach to drinking water infrastructure improvement is necessary to consistently achieve concentrations of indicator bacteria deemed safe by the WHO. The observation that drinking water quality can deteriorate between treatment and the point-of-use is consistent with studies in other parts of South America (Levy et al., 2008; Rufener et al., 2010), Asia (Eshcol et al., 2009; Kær Jensen et al., 2002), and Africa (Clasen and Bastable, 2003). In many of these studies, drinking water handling and storage at the household level was highlighted as a potential area for improvement (Noble et al., 2003). This study also explored potential sources of drinking water contamination and confirmed human carriage of the Bacteroides HF183 MST marker in the Galapagos Islands. Because the Galapagos are so geographically remote, the prevalence of these markers in the intestinal flora of archipelago residents lends credence to the idea that they will be found in various remote populations around the globe. Although validation studies should always be performed prior to use of MST assays in a new population, these findings may have implications to other geographically distinct populations affected by poor drinking water quality such as those found in many developing world countries. To our knowledge, this study is the first to use MST markers in South America to determine specific sources of fecal indicator bacteria in drinking water. The combination of conventional and alternative methods found in this study generates a more complete picture of drinking water contamination than is typically found in the literature and provides useful data for planning and remediation. Although fecal indicator bacteria were found in drinking water at the point-of-use, the lack of human-specific MST markers raises questions about the origin of observed contamination and the remediation steps that are most likely to be effective. It is not clear whether the observed E. coli contamination is truly fecal in origin or perhaps the result of environmental regrowth as has been reported previously for soil and surface waters in other tropical/subtropical regions (Fujioka and Unutoa, 2006; Toranzos, 1991). The detection of FIB in the absence of human-source markers could also suggest that fecal contamination from non-human animals is able to infiltrate the system, which would also be a health concern. Expanding the conventional and alternative analysis of drinking water to include pre- and post-storage at the household level could provide further insight to possible sources of contamination and the most appropriate steps to achieve drinking water free of E. coli. This study would have been improved by including measurements throughout the year to account for seasonal variation including changes in precipitation. Because of personnel and
funding limitations, the study could only be performed during the months of June and July in consecutive years. This time period occurs during the initial months of the dry season, which results in less rainwater runoff that may contribute to bacterial transport through the environment. Future research could strengthen the findings of this report by examining drinking water quality differences between the wet (December to May) and dry (June to November) seasons. In conclusion, this study demonstrates that drinking water handling and storage are critical in maintaining microbial water quality at points of use. Both treatment and distribution should be considered when planning water infrastructure improvements in developing and developed countries. Contamination or regrowth post-treatment likely explains much of the observed variation between sites. Improving the rate of compliance to the cleaning and maintenance schedules recommended by the municipality may improve drinking water quality. Education programs to teach the public about the importance of proper care and maintenance for household water cisterns may be useful. In the long term, a plan to construct neighborhood-level cisterns will move the maintenance and cleaning tasks from local residents to municipal employees. The shift of responsibility for daily compliance tasks may improve post-treatment drinking water handling and storage practices island-wide, thereby improving drinking water quality at the points-of-use. Finally, 24 h operation of the drinking water distribution system at pressure could create more predictable residence times, reduce the likelihood of contamination during distribution, and improve the ability to accurately dose chlorine to the system. Available water quantity may preclude this approach during certain times of the year; however, seasonal implementation may be feasible. Conflict of interest declaration The authors declare they have no actual or potential conflicts of interest. Acknowledgments The authors would like to acknowledge the support of several programs within the University of North Carolina for their support: the Jon Curtis Student Enrichment Fund - Summer Scholarship; the Center for Global Initiatives - Vimy Global Team Award; and the Carolina Center for Public Service - Hyatt Rotary Public Service Award and Sherpa Fellowship. In addition, the logistical and financial assistance provided through the Galapagos Science Center and the UNC Center for Galapagos Studies was invaluable to the completion of this project. Finally, the authors would like to thank Shannon Steel for her contributions to visualizing these data through GIS. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijheh.2017.01. 010. References ¨ Åstrom, J., Pettersson, T.J.R., Reischer, G.H., Norberg, T., Hermansson, M., 2015. Incorporating expert judgments in utility evaluation of bacteroidales qpcr assays for microbial source tracking in a drinking water source. Environ. Sci. Technol. 49, 1311–1318, http://dx.doi.org/10.1021/es504579j. Bain, R., Cronk, R., Wright, J., Yang, H., Slaymaker, T., Bartram, J., 2014. Fecal contamination of drinking-water in low- and middle-income countries: a systematic review and meta-analysis. PLoS Med. 11, http://dx.doi.org/10.1371/ journal.pmed.1001644.
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Please cite this article in press as: Gerhard, W.A., et al., Water quality at points-of-use in the Galapagos Islands. Int. J. Hyg. Environ. Health (2017), http://dx.doi.org/10.1016/j.ijheh.2017.01.010