CFDA-AM staining to assess the metabolic activity of Giardia duodenalis cysts inactivated by chlorine, boiling and ultraviolet irradiation.

Giardia duodenalis is responsible for several waterborne gastrointestinal outbreaks worldwide. In addition to limitations presented by the main disinfection methods, assessing the inactivation efficiency of cysts after the treatment also poses challenges. Thus, this study aimed to use the 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) staining protocol to evaluate the viability of G. duodenalis cysts inactivated by different UV and chlorination doses and boiling times. Under epifluorescent microscopy, metabolically active cysts that presented green fluorescence were considered viable. In contrast, when no green fluorescence could be observed, organisms were considered non-viable. Although statistical analysis revealed that increasing the UV dose did not significantly decrease the percentage of viable cysts, the fluorescence signal intensity decreased considerably when the cysts were irradiated with a dose equal to or greater than 80 mJ cm-2. Regarding chlorination and boiling treatments, this study demonstrated that no cyst showed fluorescence at the lowest NaClO concentration (0.5 mg/L) and in the shortest boiling time (2 min). Despite some limitations regarding the use of metabolic activity as a viability marker, this methodology is rapid, easy to run and cost-effective. Thus, we conclude that the CFDA-AM staining protocol has the potential to be used to assess Giardia cyst inactivation, although further research is required.

Label-free detection and enumeration of Giardia cysts in agitated suspensions using in situ microscopy.

Laboratory procedures performed in water treatment studies frequently require the characterization of (oo)cyst suspensions. Standard methods commonly used are laborious, expensive and time-consuming, besides requiring well-trained personnel to prepare samples with fluorescent staining and perform analysis under fluorescence microscopy. In this study, an easy cost-effective in situ microscope was assessed to acquire images of Giardia cysts directly from agitated suspensions without using any chemical labels or sample preparation steps. An image analysis algorithm analyzes the acquired images, and automatically enumerates and provides morphological information of cysts within 10 min. The proposed system was evaluated at different cyst concentrations, achieving a limit of detection of ~30 cysts/mL. The proposed system overcomes cost, time and labor demands by standard methods and has the potential to be an alternative technique for the characterization of Giardia cyst suspensions in resource-limited facilities, since it is independent of experts and free of consumables.

Household slow sand filters in continuous and intermittent flows and their efficiency in microorganism's removal from river water.

This study aimed to evaluate the efficiency of four household slow sand filter (HSSF) models for the removal of microorganisms from river water throughout the development of their biological layers (schmutzdecke). Two models were designed to be operated continuously (HSSF-CC and HSSF-CT) and two intermittently (HSSF-ID and HSSF-IF). Filters were fed daily with 48 L pre-treated river water (24 h sedimentation followed by filtration through a non-woven synthetic blanket). Water samples were quantified by coliform group bacteria and analysed by bright field microscopy to visualize the microorganisms. Total coliform reduction was between 1.42 ± 0.59 log and 2.96 ± 0.58 log, with continuous models showing a better performance (p-values < 0.004). Escherichia coli reduction varied from 1.49 ± 0.58 log to 2.09 ± 0.66 log and HSSF-IF, HSSF-CC and HSSF-CT presented a similar performance (p-values > 0.06), slightly better than the one presented by HSSF-ID (p-value=0.04). Microorganisms, such as algae, protozoa and helminths were detected by microscopy in raw water and pre-treated water. Algae were the most significant group in these samples, although they were not visualized by bright field microscopy in the filtered water. Results showed the potential of HSSF in microbiological risk reduction from river water, which increases the range of point-of-use water treatments in rural communities. However, additional studies of the HSSF biological layer must be performed.

Analytical challenges and perspectives of assessing viability of Giardia muris cysts and Cryptosporidium parvum oocysts by live/dead simultaneous staining.

Giardia and Cryptosporidium are pathogenic protozoa often present in the environment in their infective form(cysts and oocysts). These parasites are very resistant to disinfection, which makes them important target organisms in environmental quality monitoring and sanitation. Viability assessment provides an interpretation of cell inactivation, and it can be evaluated by membrane integrity as well as enzyme activity, using different staining methods. These are straightforward and adequate to laboratories that lack infrastructure for molecular-based technologies or animal infectivity tests. This study investigated simultaneous staining by a commercial live/dead kit, in order to assess viability of Cryptosporidium parvum oocysts and Giardia muris cysts, comparing it to propidium iodide (PI) incorporation, a common stain applied in viability estimation. Results suggested that, although the central hypothesis of one-panel visualization (α = 0.05) was met, simultaneous staining impaired (oo)cyst detection by immunofluorescence assay (IFA), which was found to be essential to enumeration, as the live/dead test led to poor (oo)cyst labelling or a 10-fold lower recovery when carried out concomitantly to IFA. As for the viability assessment itself, although red dye uptake occurred as expected by dead or weakened organisms, neither live G. muris cysts or C. parvum oocysts present any green fluorescence by esterase metabolism. This may have been caused by low enzyme activity in the infective form and/or wall thickness of these parasites. The results do not exclude the possibility of simultaneous fluorescence staining for protozoa, but it is a starting point for a broader analysis, that may consider, for instance, different incubation conditions.

Evaluation of a multi-barrier household system as an alternative to surface water treatment with microbiological risks.

Household Water Treatment and Safe Storage (HWTS) are recommended to supply the demand for drinking water in communities without conventional water supply systems. However, there is a lack of long-term laboratory studies regarding such technologies. We evaluated the contributions of each step of a multi-barrier system with pretreatment (sedimentation and fabric filtration), filtration in Household Slow Sand Filters (HSSFs) and disinfection (sodium hypochlorite) treating surface water for more than 14 consecutive months. Removal of turbidity, colour, organic matter, coliform group bacteria and protozoa were evaluated. Two HSSF models were compared, one with a diffuser vessel (HSSF-d) and one with a gravity float equipped vessel (HSSF-f). Correlations between efficiency and operational parameters were assessed. Overall, the multi-barrier system removed more than 90% of turbidity and more than 3.5 log of Escherichia coli. HSSF removed up to 3.0 log of Giardia spp. and 2.4 log of Cryptosporidium spp. HSSF-f presented significantly higher removal rates for turbidity, apparent colour and E. coli. Disinfection resulted in water with E. coli concentration lower than 1 CFU 100mL-1, however it was not able to inactivate protozoa. The evaluated system was able to reduce microbiological risks from water and could indeed be an alternative to communities that depend on surface water as their main source of supply. Nevertheless, further studies are recommended to include a low-cost disinfectant for protozoa inactivation.

Household slow sand filter efficiency with schmutzdecke evaluation by microsensors.

Slow sand filtration is a common technology providing potable water in rural households across Latin America, Asia and Africa. Two PVC household slow sand filters (HSSF) were operated in continuous (C-HSSF) and intermittent (I-HSSF) flow modes for eight consecutive months. A non-woven blanket was installed on the fine sand top to facilitate cleaning with scheduled maintenance undertaken every 30 days. The efficiency of each HSSF was evaluated via physico-chemical indicators (reduction of turbidity and colour) with biological performance assessed via total coliform and E. coli enumeration post treatment. There were no statistically significant differences between the continuous flow and intermittent flow models for physical-chemical and total coliform reduction parameters. However, when evaluating E. coli, C-HSSF performed better (p = 0.02). The non-woven blanket was subjected to weekly analysis using a Clark-type amperometric microsensor (diameter < 20 µm), which measured dissolved oxygen (DO) concentration in the adherent biofilm. DO microprofiles illustrated a variation in biofilm growth, which were associated with a progressive increase in the HSSF efficiency. The maximum DO depletion value measured during several months of operation showed no significant difference between I-HSSF and C-HSSF (p = 0.98). The microsensor measurements provided unprecedented results in real time. These results can help to understand the efficiency of the filter in relation to the biofilm growth, the dissolved oxygen depletion and turbidity removal.

Conceptualising global water challenges: A transdisciplinary approach for understanding different discourses in sustainable development.

Global challenges impact upon substantial numbers of people in different locations and inform policy at multiple levels under the United Nation's Sustainable Development Goals (SDGs). An aspect of the SDGs framework is the stated inter-relationship between SDGs and local, regional and global partnerships for research and development. In response to dissatisfaction with existing approaches to addressing such complex problems the purpose of this paper is to propose a problem-language-context (PLC) model as a way of framing sustainable development challenges; and in so doing create a heuristic that allows challenges such as water security to be understood using a logically consistent framework. Such an approach builds on a growing transdisciplinary innovation literature that strives to generate knowledge that is problem-focused and inclusive of both scientific and societal stakeholders. The utility of the PLC model is then examined using a case study review carried out on a body of evidence - the United Nations World Water Development Reports (WWDRs) 2003-2019. The result of this review suggests that such problem framing can be of value in revealing the implicit (and sometimes contradictory) assumptions held by policy makers, practitioners and researchers. The main conclusion is that a transdisciplinary approach is one way of better understanding some of the conflicting viewpoints evident in discipline-based approaches to sustainable development, global water challenges and water security.

Does each bead count? A reduced-cost approach for recovering waterborne protozoa from challenge water using immunomagnetic separation.

Giardia duodenalis and Cryptosporidium spp. are two of the most prominent aetiological agents of waterborne diseases. Therefore, efficient and affordable methodologies for identifying and quantifying these parasites in water are increasingly necessary. USEPA Method 1623.1 is a widely used and validated protocol for detecting these parasites in water samples. It consists of a concentration step, followed by parasite purification and visualization by immunofluorescence microscopy. Although efficient, this method has a high cost particularly due to the immunomagnetic separation (IMS) step, which is most needed with complex and highly contaminated samples. Based on this, the present study aimed to determine whether it is possible to maintain the efficiency of Method 1623.1 while reducing the amount of beads per reaction, using as a matrix the challenge water recommended by the World Health Organization. As for Giardia cysts, a satisfactory recovery efficiency (RE) was obtained using 50% less IMS beads. This was evaluated both with a commercial cyst suspension (56.1% recovery) and an analytical quality assessment (47.5% recovery). Although RE rates obtained for Cryptosporidium parvum did not meet Method 1623.1 criteria in any of the experimental conditions tested, results presented in this paper indicated the relevance of the described adaptations, even in challenge water.

Filter media depth and its effect on the efficiency of Household Slow Sand Filter in continuous flow.

This study evaluated the impact of a 50% reduction of filter media depth in Household Slow Sand Filters (HSSFs) on continuous flow to remove physicochemical and microbiological parameters from river water. Furthermore, simple pre-treatment and disinfection processes were evaluated as additional treatments. Two filter models with different filtration layer depths were evaluated: a traditional one with 50 cm media depth (T-HSSF) and a compact one (C-HSSF) with 25 cm. HSSFs were fed with pre-treated river water (24-h water sedimentation followed by synthetic fabric filtration) for 436 days at a constant filtration rate of 0.90 m3 m-2 day-1 with a daily production of 48 L day-1. Sodium hypochlorite (2.0 mg L-1 of NaOCl 2.5% for 30 min) was used to disinfect the filtered water. Water samples were analysed weekly for parameters such as turbidity, organic matter, colour and E. coli, among others. Removal of protozoan cysts and oocysts by the HSSFs were also evaluated. After pretreatment, turbidity from the HSSF river water was reduced to 13.2 ± 14.6 NTU, allowing the filters to operate. Statistical analysis indicated no significant difference (p > 0.05) between T-HSSF and C-HSSF efficiencies in all evaluated parameters throughout operation time. Hence, media depth reduction did not hinder continuous HSSF performance for almost all the evaluated parameters. However, it may have affected Giardia cysts retaining, which passed through the thinner media on one evaluation day. Disinfection was effective in reducing remaining bacteria from filtered water; however, it was ineffective to inactivate protozoa. The reduction in the filtration layer did not affect the overall filtered water quality or quantity showing that a compact HSSF model may be a viable option for decentralized water treatment.

Detection of Giardia and Cryptosporidium in environmental matrices with immunomagnetic separation: two or three acid dissociations.

This study evaluated the technology of detection of Giardia spp. cysts and Cryptosporidium spp. oocysts in environmental matrices obtained after water treatment on a bench scale. Calcium carbonate flocculation with immunomagnetic separation was the selected method to quantify the protozoa, and the importance of the number of acid dissociations in the immunomagnetic separation was assessed. When adding the third acid dissociation, an increase of 71% ± 6 in floated residue and 31.9% ± 28.7 in filter backwash water in cyst recovery was observed, while in oocyst recovery, a non-significant increase was detected. In the filtered water, this increased dissociation was important in the protozoa recovery with increases greater than 33%. The results showed that there is a strong interaction of these target organisms with the magnetic microspheres, since protozoa were still recovered in the third acid dissociation and some of them were still adhered to the magnetic microspheres.

Giardia spp. cysts and Cryptosporidium spp. oocysts in drinking water treatment residues: comparison of recovery methods for quantity assessment.

Water treatment plant (WTP) residues, e.g. sludge and filter backwash water (FBW), may contain pathogenic microorganisms, as Giardia spp. and Cryptosporidium spp. However, recovering protozoa from such matrices lacks a formal and precise protocol, which is imperative to improve research in their detection, removal and inactivation. The latter includes a deeper challenge as some recovery methods may compromise viability. This study applied different recovery methods for G. muris cysts and C. parvum oocysts spiked into settled sludge and FBW obtained from a bench treatment. Procedures in sludge involved direct centrifugation, alkaline and acid flocculation, including purification by immunomagnetic separation (IMS). FBW samples were tested for membrane filtration (MF) and heated Tween® scrapings followed or not by IMS. Propidium iodide (PI) inclusion was used for oocyst viability evaluation prior and after recovery. Results with purified suspensions lead to higher recovery efficiencies (RE) for C. parvum, which was assumed to relate to poor G. muris fluorescence. Analytical quality assessments were carried out with ColorSeed® for the methods that stood out for each matrix and the results indicated lower RE than when organisms from purified suspensions were recovered. Ferric sulphate flocculation and MF, both followed by IMS reached 32.25% and 11.00% RE for Giardia spp. and 19.61% and 2.00% for Cryptosporidium spp., respectively. All of the tested methods affected oocyst viability. These results encourage further research to overcome the matrices complexity explained in this paper and increase RE, taking effects in protozoa viability into consideration.

Cryptosporidium spp. and Giardia spp. (oo)cysts as target-organisms in sanitation and environmental monitoring: A review in microscopy-based viability assays.

Cysts and (oo)cysts are the infective forms of parasitic protozoa, as Giardia and Cryptosporidium, which are widespread and associated to worldwide waterborne diseases outbreaks. These microorganisms pose a challenge to public health, as they are resistant to conventional disinfection methods, which make them important parameters when evaluating inactivation efficiency. However, when (oo)cysts are targets, it is challenging to infer inactivation efficacy, as it may require infectivity tests that are not often an option for laboratory routine analysis. In this scene, (oo)cyst viability based on induced excystation, membrane integrity and enzyme activity evaluated by dye inclusion and/or exclusion, as well as fluorescence reduction consist on microscopy-based techniques that may be options to estimate inactivation in the environmental context. This scoping review presents applications, advantages and limitations of these methodologies for viability assessment, in order to shed light on the (oo)cyst viability topic and provide insight strategies for choosing protocols in the environmental and sanitation field, in laboratory applications and novel research.

Pretreatment using Opuntia cochenillifera followed by household slow sand filters: technological alternatives for supplying isolated communities.

Household slow sand filter (HSSF) performance in continuous and intermittent flows was evaluated when influent water was treated with a natural coagulant extracted from Opuntia cochenillifera. The water under study, used as influent, had a turbidity of 111 ± 17.3 NTU. When clarifying the water with O. cochenillifera, the best condition obtained was 30  mg L-1 in natural pH (without correction), generating clarified water with turbidity satisfactory to filters operation (7.83 ± 2.32 NTU). The results indicated a better performance of continuous flow HSSF in turbidity removal (79.2% ± 8.39%) and higher efficiency of intermittent flow HSSF in the removal of E. coli (2.86 log ± 0.79 log for 12  h pause period and 2.41 log ± 0.42 log for 4  h pause period). For the sake of comparison, the evaluated HSSFs had the same production (60 L day-1). The impact on the interruption of the 96-h feed into the HSSFs was analysed and the results indicated a significant change in the quality of the filtered water after resuming the operation. This fragility of technology must be considered when it is implemented as lack of water can be a reality in the target communities. Acute toxicological assays with C. xanthus larvae showed no toxicity for pretreated and filtered water; however, more testing should be performed.

Household slow sand filters with and without water level control: continuous and intermittent flow efficiencies.

Four household slow sand filters were made out of PVC and operated in continuous and intermittent flows, with and without using a float to control the maximum level of water inside the units. The efficiency was evaluated as a function of Escherichia coli reduction and turbidity in water from the study prepared with kaolinite and E. coli suspension. The correlation of the efficiencies with the following operational parameters was evaluated: operating time, time after maintenance, filtration rate and head loss divided by bed thickness. The filters were classified as intermittent with float (IFF), intermittent without float (IF), continuous with float (CFF) and continuous without float (CF). IFF, CFF and CF had a non-woven blanket installed on top of the media. The results indicated that no significant statistical differences were found in E. coli reduction and turbidity between IFF and IF, however the former had filter runs over 80 days and the latter almost a quarter of this value. CFF matured faster and had less turbidity remaining in relation to CF. When comparing IFF with CFF, the former presented lower turbidity remaining (0.89 ± 0.44 NTU versus 1.24 ± 0.91NTU), but a lower reduction of E. coli (1.40 ± 0.61 log versus 2.29 ± 0.74 log). The time after maintenance was the most important operational parameter when evaluating the efficiencies. The float helped to mature the filter more quickly in a continuous flow and, together with a non-woven blanket, extended the filter runs in the intermittent flow.

Household slow sand filters in intermittent and continuous flows to treat water containing low mineral ion concentrations and Bisphenol A.

Household slow sand filter (HSSF) has been used as an alternative to drinking water treatment in rural communities worldwide; however, its performance to treat influent water with quality similar to rainwater still needs further studies. Rainwater presents low pH and slight mineral ion concentrations, an aspect that can modify the filter media and consequently the HSSF efficiency. Furthermore, house roofs used in rainwater harvesting can be made of plastic. Therefore, it can introduce chemicals such as Bisphenol A (BPA) in the water. In this context, two pilot-scale HSSFs operated in continuous and intermittent flows were evaluated to treat water containing BPA and low mineral ion concentrations in order to assess the filter performance. Filter media leaching was noticed in the trials; thus, filter media and construction material selection must be carefully evaluated to eliminate risks of pollutant occurrence in drinking water. Operational differences between continuous and intermittent flows influenced the HSSF efficiency for BPA and DOC removals; even so, the filters' performance was low probably due to the slow schmutzdecke development. According to tracer test results, HSSF can be classified as a plug flow reactor and strategies to improve its hydraulic performance are not required.

Coagulation, flocculation, dissolved air flotation and filtration in the removal of Giardia spp. and Cryptosporidium spp. from water supply.

Removing protozoa from a water supply using coagulation, flocculation, dissolved air flotation (DAF) and filtration on a bench scale was evaluated. Calcium carbonate flocculation with and without immunomagnetic separation (IMS) was chosen to detect Giardia spp. cysts and Cryptosporidium spp. oocysts in the studied samples. The results indicated that DAF removed between 1.31 log and 1.79 log of cysts and between 1.08 log and 1.42 log of oocysts. The performance was lower in filtration, with the removal of 1.07 log-1.44 log for cysts and 0.82 log-0.98 log for oocysts. The coagulation, flocculation, DAF and filtration steps removed more than 2.2 log of cysts and oocysts from the water studied. However, protozoa were detected in the filtered water, even with turbidity values of 0.2 NTU. The recovery of the detection method met the international criteria and was higher when there was no IMS. Including the third acid dissociation in the IMS was critical to improve the performance of the protocol tested. However, there was an increase in the technical and analytical complexity and costs. It was also observed that the efficiency of the treatment was linked to the performance of the selected method of detecting protozoa.

Performance comparison of three methods for detection of Giardia spp. cysts and Cryptosporidium spp. oocysts in drinking-water treatment sludge.

Detecting pathogenic protozoa in drinking-water treatment sludge is a challenge as existing methods are complex, and unfortunately, there are no specific technical standards to follow. Selecting an efficient analytical method is imperative in developing countries, such as Brazil, in order to evaluate the risk of parasite infection. In this context, three methods to detect Giardia spp. cysts and Cryptosporidium spp. oocysts were tested in sludge generated when water with protozoa and high turbidity was treated. Jar testing was carried out using polyaluminium chloride as a coagulant to generate the residue to be analyzed. The results showed that calcium carbonate flocculation with reduced centrifugation and immunomagnetic separation obtained the highest recoveries in the tested matrix showing 60.2% ± 26.2 for oocysts and 46.1% ± 5 for cysts. The other two methods, the first using the ICN 7× cleaning solution and the second considering the acidification of the sample, both followed by the immunomagnetic separation step, also presented high recoveries showing 41.2% ± 43.3 and 37.9% ± 52.9 for oocysts and 11.5% ± 85.5 and 26% ± 16.3 for cysts, respectively. Evidently, these methods and others should be studied in order to make it possible to detect protozoa in settled residue.

Uso de modelação matemática para projeto de câmaras mecanizadas de floculação em série em estações de tratamento de água / The use of mathematical modeling to the design of continous flow completely mixed chambers in series for flocculation in water treatment plants

A metodologia encontrada na literatura para a otimização do gradiente de velocidade médio em reatores de mistura completa, com câmaras em série de escoamento contínuo, a partir de ensaios em reator estático tipo jarteste, baseia-se em modelos matemáticos que descrevem a cinética dos encontros entre partículas durante a floculação. Considerando-se que a aplicabilidade desta metodologia tem-se revelado inconsistente, como observado por diversos autores, este trabalho propõe uma metodologia racional para utilizar e otimizar os dados experimentais obtidos em reatores estáticos e, utilizando modelos matemáticos, obter dados para o projeto de floculadores mecanizados em série em estações de tratamento de água.