TweetAbstract This paper provides a followup on the health impact of providing access to water treatment and flush toilets to region of Honduras. Significant reductions were found in the one-year incidence of positive test results for the three protozoan species tested. This finding combined with the previously reported ethnographic and medical chart review data provides […]
This paper provides a followup on the health impact of providing access to water treatment and flush toilets to region of Honduras. Significant reductions were found in the one-year incidence of positive test results for the three protozoan species tested. This finding combined with the previously reported ethnographic and medical chart review data provides compelling evidence that such interventions significantly reduce the disease load from waterborne pathogens within this population. Furthermore, the finding that initial results are significantly different, even in the initial round of testing, if individuals who are not followed up are eliminated from the analysis has profound methodological implications which warrant further investigation and demonstrates the need for precise definitions of community in future studies.
A key component of the United Nations Millennium Development Goal Number 7 states “halve, by 2015 the proportion of the population (global) without sustainable access to safe drinking water and basic sanitation.” Most waterborne diseases result in diarrhea which continues to be a leading cause of morbidity and mortality worldwide. According to World Health Organization data, using existing technologies approximately ten percent of the worldwide burden of disease would be removed by the water supply, sanitation, hygiene, and management of water resources, making water-related diseases arguably the most manageable set of health problems affecting humans .
A great deal of work has been done attempting to measure the impact of interventions to provide improved water sources at the household level, and less frequently at the community level. The overwhelming majority of these studies have also used either key informant or self-reporting of diarrhea (defined as three or more loose stools per day) as the measure of disease burden [2–4]. Reliance upon such nonobjective measures introduces a host of potentially confounding variables [2, 5, 6] and yet appears to have been used in all of the 2,120 published studies reviewed in a far reaching meta-analysis produced for the World Bank on diarrhea and water interventions . While some of these deficiencies may be reduced by shortening recall time to seventy-two hours or less, potentially profound observer effects remain. Estimates of disease load changes are further impeded by researchers’ concentration on known users of water systems rather than measurements on community disease levels of disease changes regardless of compliance, thus making extrapolations of disease rate changes inappropriate.
In 2006, Water Missions International (WMI) received a grant from the Pentair Foundation to provide improved water source access and toilet systems to all of the people in the district of Colon, Honduras, an area that contains approximately 340,000 people. The goal of 100% coverage utilizes a combination of solutions including home-based filtration systems (provided by a different NGO) for communities with less than 300 people, and a variety of high-capacity treatment systems for larger communities. In the initial phase of the study, all 604 water sources for the control and test communities were tested by pressure filtration methods, and all failed quality testing by being positive for coliform bacterial growth. For all households that lacked adequate sanitation facilities and agreed to assist in installation, sanitary pit latrines with pour-flush toilet (toilets with water traps and no reservoir that are flushed by pouring a bucket of water into the basin) were also provided. Water treatment systems and pour-flush toilets deployed during the present study were developed and manufactured by Water Missions International, a nonprofit organization based in Charleston, SC, that works to provide sustainable water treatment capacities and sanitation facilities for people in developing countries. The technology uses a combination of multimedia, multistage filters, and chlorination to provide treated water for drinking and cooking that meets the most stringent class of WHO drinking water standards as well as passed present US standards from the EPA—specifically, tests on treated water grew no coliform bacteria on repeated, monthly testing during the period of this study. In addition, WMI provides community development programs that include education and microenterprise strategies to assure sustainability of these interventions.
In the baseline study phase of the project, water sources for 613 communities were identified. Various water quality tests were conducted on the community water sources by standard membrane filter test. High counts of coliform bacteria indicative of fecal contamination were found in 100% of the water sources. As previously published , initial stool tests also showed that 29.3% (53 of 181) of the volunteer subjects from the twelve communities that had been randomly selected from the state of Colon carried at least one of the three tested protozoan parasites. Also as reported previously, the prevalence of positive protozoan parasite levels was significantly lower in the intervention groups as compared to the test group. Even greater reductions of disease rates (at least 52%) were noted in the medical chart reviews of visits to the local health facility for diarrhea and dysentery as well as self-reporting of the same diseases via ethnographic interviews. Ethnographic data further suggested widespread acceptance and community-wide reproduction of the awareness of health benefits derived from consuming treated water. The present paper is a followup to that report primarily looking at the development of positive parasite stool tests in the same communities twelve months after the initial round of tests with an expanded study population.
Twelve communities from three different categories were randomly selected from the Colon district. Four communities had not yet obtained a water treatment system and were used collectively as the Control Group. Four other communities where water systems (Water Only Group) had been deployed were entered into the study as were four more communities where both water systems and sanitary flush latrines had been installed (Water and Sanitation Group). For both the initial tests in 2009 and the final round of tests in 2010, volunteers were recruited by poster advertising and via community leaders. Methods of recruitment were identical in all communities. All subjects who tested positive for protozoan antigens in either study (2009 or 2010) were treated with an appropriate dose of tinidazole (500 mgs per day for three days for adults with weight adjusted dosing for children). Subjects were directly observed to take the initial dose, which in management of Giardia has been shown to be highly effective without the additional doses. No side effects of treatment were reported. One hundred and sixty-three of the 200 subjects in the final round of testing in 2010 had also participated in the prior studies conducted 12 months earlier.
The effect of previous medical treatment of subjects upon the present study is to provide a population that either tested negative or were given highly effective treatment in 2009—thus providing a subpopulation which was believed to have begun the 12-month test period free of any of the three tested protozoans. The results among the subjects tested both times, therefore, may be regarded as the rates of reinfection over the 12-month period (or a one-year incidence rate) for all three groups.
Recent advances in highly sensitive and specific rapid immunoassays for waterborne parasite diseases have made field testing of individual fecal specimens now possible [9, 10]. These devices test for species-specific antigens of common parasites known to be primarily waterborne. The device chosen for this study tested for three protozoan parasites: Giardia lamblia (now widely known as Giardia intestinalis), Entamoeba histolytica/Entamoeba dispar, and Cryptosporidium parvum antigens. Previous work has shown these tests to have both specificity and sensitivity in excess of 96% for the aforementioned pathogens.
Immunoassay of stool for these waterborne parasites was used as an indicator that the subject had been exposed to waterborne pathogens and was therefore at risk of these and other infectious waterborne illnesses. A separate subset of 163 subjects from the three groups who also gave specimens twelve months earlier was analyzed as a separate subgroup. Given the highly effective cure rates of tinidazole for Giardia and Entameba and the fact that the vast majority of non-immunocompromised subjects will clear Cryptosporidium infections spontaneously, this subset is thought to represent recolonization rates with waterborne protozoan during the year after the initial round of testing and treatment. All specimens were tested within 12 hours of collection using the Triage Micro Parasite Panel manufactured by Biosite Incorporated.
Further information regarding diarrhea and dysentery rates was obtained by reviewing medical records from a public health clinic in a community where a water treatment system had been previously installed. Ethnographic data was collected using a combination of KAP surveys and guided interviews. The medical records review and the majority of the ethnographic data have been previously reported in this journal .
3. Role of the Funding Source and Ethics Review
Water Missions International maintains a country program in Honduras whose staff provided support and significant amount of labor for this project. The study design, collection, and analysis of data and interpretation of the data were the sole responsibility of the author.
Prior to initiation of the study, the Colon Minister of Health and the Institutional Review Board of Water Missions International reviewed and gave approval and consent for the project and study. Consistent with this review, no information from medical chart reviews which could identify subjects of the study was retained outside of the local healthcare facility. Under the supervision of a licensed physician, all individuals in whom potential pathologic parasites were found were given free treatment with regimens previously approved by the Colon Minister of Health. The control communities where no water treatment or sanitation facilities existed were selected from a preexisting construction queue and intervention was not withheld as a result of this study. Verbal consent was obtained and recorded from all subjects.
Age distributions within the three groups are seen in Tables 1 and 2. Gender distribution is seen in Table 3. Parasite test results for the control group compared to the combined water only group and water and sanitation group are seen in Table 4. Giardia and Entameba accounted for all but one positive test in all categories. Giardia accounted for 46% and Entameba 48% of the positive tests while Cryptosporidium remained rare at 6% of the totals.
Table 1: Age distribution of all subjects in all groups.
Table 2: Mean age distribution by group.
Table 3: Gender by intervention group.
Table 4: 2010 data comparing control group to the combination of water only group and water and sanitation group.
Subjects living in communities that did not have access to water systems had significantly higher rates of positive tests than subjects who had either access to water or who had access to water and flush toilets both in the initial survey of 2009 and in the 2010 followup (). These finding are summarized in Figure 1.
In 2009, a comparison of the rate of positive parasite tests appeared to demonstrate that, while access to a water treatment system reduced parasite levels, communities that had both treatment systems and installed flush toilets demonstrated a higher rate of positive parasite tests. These findings show a distinct gender bias toward women and are summarized in Figure 2. Additional ethnographic witnessing suggested that this finding may possibly be explained by the fact that women exclusively cleaned the toilets, often with inadequate supplies and protection. Water Missions International responded to this suggestion with additional training and supplies. In the followup parasite survey of 2010, what had appeared to have been a negative effect of the toilet systems was no longer present, as seen in Figure 2.
A separate analysis of parasite test results including only subjects who were available in both 2009 and 2010 is summarized in Figure 3. Of interest is that the apparent negative effect of the toilets in the 2009 data (Figure 1) completely vanishes when subjects lost to follow up in 2010 are removed from the 2009 analysis.
The present paper is a followup of the research previously reported  and adds support to the conclusion that access to community-based water treatment systems and flush toilets reduced the disease load in this region of Honduras. To our knowledge, these are the first studies to combine self-reported data, medical chart review, and stool immunoassays as an indicator of exposure to potential waterborne pathogens. The triangulation of these methodologies provides powerful support to what are otherwise strongly subjective and questionable measures of disease loads from waterborne pathogens.
The previously reported ethnographic data from these communities suggests a high level of understanding of the causes and prevention of diarrhea among the communities studied. The overwhelming majority (130 of 142) of the people interviewed attributed the majority of their diarrheal diseases to water and sanitation issues and improvement of the condition to improved water sources and access to flush toilets. There were also significant signs of a shift of ideations regarding drinking untreated water toward an appreciation of the importance of purified water and prohibitions against drinking untreated water with the addition of water treatment facilities and community education.
Ethnographic data found during this study suggests that, consistent with other similar work, the availability of improved water is felt by its recipients to improve a general sense of health and well-being. High levels of knowledge related to water issues exist in this area of Honduras which could be attributed to many factors including sophisticated public health efforts, high literacy rates comparable to the region, widespread health education in public schools, and the training offered by Water Missions International and other NGOs. Local indigenous belief systems appear uncommonly (mentioned in only 6 of 46 individual interviews and in none of the four focus groups) and for no one were they the basis for a preferred method of treatment.
Immunoassay evidence of decreased prevalence of waterborne parasites strongly supports the contention that community-based water treatment facilities reduce the overall stool parasite load, at least of the three protozoan species tested. Since all subjects who tested positive for either Giardia or Entameba were treated with three doses of tinidazole adjusted for age and weight, the follow-up study of all subjects who submitted stool samples initially is felt to represent the reinfection rates, in essence eliminating concerns regarding residual colonization from exposures that occurred prior to initiation of this study. Previous treatment of subjects who tested positive for any parasite creates the potential for a Hawthorne effect; however, the elevated number of positive tests initially found within the control communities would potentially bias this group more than the test groups and would tend to lessen rather than strengthen the differences found.
Also, as previously reported , when parasite antigens were detected in stool samples of individuals who had access to improved water sources, ethnographic investigation revealed lapses of behavior in spite of the high level of understanding of the risks associated with drinking from untreated sources. Further analysis of the interviews of subjects whose stool was positive for potential waterborne parasites suggests that risk and time management decisions rather than cultural or knowledge-based differences accounted for lapses in behavior and willingness to drink untreated water. Subjects reported that the time required to obtain treated water, sometimes a difference of only a minute or less, was too great to overcome their concerns with potential health risks associated with untreated tap water.
Multiple pathogens and inflammatory conditions cause diarrhea, making monitoring this symptom alone an inexact measure of the disease load related to water quality. Worldwide, the most common causes of diarrhea are viral infections such as the rotavirus, an ubiquitous infection that may be transmitted by personal contact. Food contamination and noninfectious inflammatory diseases add to the diarrhea prevalence. Though not precisely known, the number of diarrhea cases unrelated to waterborne pathogens is likely substantial. This means that the 52% drop in diarrhea rates noted in the previously reported community chart reviews may represent an even greater majority of the cases that could possibly be related to potential water and sanitation issues. This follow-up study strongly supports this finding and suggests that the effect where water treatment systems are maintained may even increase over time.
A comparison of the 2009 parasite test data excluding those lost to follow up a year later was dramatically different from when these individual tests were included (Figure 3). This suggests that the population that was lost to follow up significantly added to the initial rate of positive tests. This phenomenon deserves future scrutiny and incorporation into discussions of the idea of community as a social construct. If community is defined as those present at a given point in time, we see a negative impact from presence of the toilets. When we define community as those who reside in the geographic area for at least one year we find the exact opposite as this apparent negative impact is not detected. As this finding demonstrates, the unit of analysis remains paramount in such studies.
This combination of qualitative data, health records reviews, and immunoassays provides compelling evidence that community-based water treatment facilities with or without providing flush toilets significantly reduced the burden of diseases in the communities of Colon, Honduras. We further validate with objective measures prior work based upon self-reporting of diarrhea rates. Finally, this data suggests that interventions to provide potable water access on a community level when combined with community development efforts and sanitation can play a significant role in the reduction of mortality and morbidity from waterborne diseases and associated comorbidities.
Providing access to water treatment or water treatment and flush toilets significantly reduced the one-year incidence of positive test results for the three protozoan species tested. This finding combined with the previously reported ethnographic and medical chart review data provides compelling evidence that such interventions significantly reduce the disease load from waterborne pathogens within this population. Furthermore, the finding that initial results are significantly different, even in the initial round of testing, if individuals who are not followed up are eliminated from the analysis has profound methodological implications which warrant further investigation.
Adding a temporal definition of community resulted in a completely different finding regarding the impact of supplying flush toilets, demonstrating the need for precise definitions of community in future studies.
The method used here where objective measurements of health effects are coupled with more traditional anthropological tools may serve as a template for future studies in medical anthropology.
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by Jeffery Deal
Health Studies at Water Missions International, 2049 Savannah Highway, Charleston, SC 29407, USA
Received 1 November 2011; Accepted 30 November 2011
Academic Editor: Kaushik Bose
Copyright © 2011 Jeffery Deal. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Tweet Myron l Meters Ultrameter III 9PTKA from Myron L Meters
TweetThe PT1 is designed to be very reliable and requires only infrequent calibration. Myron L Meters recommends calibrating each measurement mode you use once monthly. However, you should check the calibration whenever measurements are not as expected. The PT1 is programmed for 2 calibration options: Wet Calibration or Factory Calibration. Wet calibration is most accurate. […]
The PT1 is designed to be very reliable and requires only infrequent calibration. Myron L Meters recommends calibrating each measurement mode you use once monthly. However, you should check the calibration whenever measurements are not as expected. The PT1 is programmed for 2 calibration options: Wet Calibration or Factory Calibration. Wet calibration is most accurate. But if a high quality standard KCl-1800 µS or 442-3000 ppm solution is not available, the PT1 can be returned to factory settings.
Use calibration solution specified for measurement mode: Use KCL- 1800 for Cond KCl; Use 442-3000 for tdS 442, SALt 442, tdS NaCl, and SALt NaCl. See Specifications table for 442 solution ppm NaCl equivalent value. Calibrating TDS simultaneously calibrates SALt for the same value and vice versa.
1. Pour calibration solution into a clean container.
2. Rinse the pen 3 times by submerging the cell in fresh calibration solution and swirling it around.
3. Remove pen from solution, then fill the container one more time.
4. Press and release the push button. The LCD will briefly display the firmware version then the current measurement mode. Ensure the PT1 is in the correct solution mode.
5. Immediately push and hold the push button. The display will scroll through “CAL”, “SOL SEL”, “FAC CAL”, “ºCºF TEMP”, and “ESC”. Release the button when “CAL” displays.
6. Grasp the pen by its case with your fingers positioned between the
display and the pen cap to avoid sample contamination.
7. While the LED flashes rapidly, dip the pen in calibration solution so that the cell is completely submerged. If you do not submerge the cell in solution before the flashing slows, allow the pen to power off and start over.
8. While the LED flashes slowly, swirl the pen around to remove bubbles, keeping the cell submerged. Keep pen at least 1 inch (2½ cm) away from sides/bottom of container.
9. When the LED light stays on solid, remove the pen from the solution. “CAL SAVED” will display indicating a successful calibration.
Note: If an incorrect solution is used or the measurement is NOT within calibration limits for any other reason, “Error” displays alternately with “CLEAn CEL/CHEC SOL”. Check to make sure you are using the correct calibration solution. If the solution is correct, clean the cell by submerging the cell in a 1:1 solution of Lime-A-Way® and water for 5 minutes. Rinse the cell and start over.
10. Small bubbles trapped in the cell can give a false calibration. Measure the calibration solution again to verify correct calibration. If the reading is not within ±1% of the calibration solution value, repeat calibration.
If you do not have the proper calibration solution or wish to restore the pen to its original factory settings for any other reason, use the FAC CAL function to calibrate the PT1.
1. Press and release the push button. The LCD will briefly display the firmware version then the current measurement mode.
2. Immediately push and hold the push button. The display will scroll
through “CAL”, “SOL SEL”, “FAC CAL”, “ºCºF TEMP”, and “ESC”. Release
the button when “FAC CAL” displays.
3. While the display scrolls through “PUSHnHLD” and “FAC CAL”, push and hold the push button until the display scrolls through “SAVEd” and “FAC CAL”, indicating the pen has been reset to its factory calibration.
4. Allow the pen to time out to turn power off.
TweetRedox potential has been identified by the Organisation for Economic Co-operation and Development (OECD) as one of the parameters that should be investigated for the testing of manufactured nanomaterials. There is still some ambiguity concerning this parameter, i.e., as to what and how to measure, particularly when in a nanoecotoxicological context. In this study the […]
Redox potential has been identified by the Organisation for Economic Co-operation and Development (OECD) as one of the parameters that should be investigated for the testing of manufactured nanomaterials. There is still some ambiguity concerning this parameter, i.e., as to what and how to measure, particularly when in a nanoecotoxicological context. In this study the redox potentials of six nanomaterials (either zinc oxide (ZnO) or cerium oxide (CeO2)) dispersions were measured using an oxidation-reduction potential (ORP) electrode probe. The particles under testing differed in terms of their particle size and dispersion stability in deionised water and in various ecotox media. The ORP values of the various dispersions and how they fluctuate relative to each other are discussed. Results show that the ORP values are mainly governed by the type of liquid media employed, with little contributions from the nanoparticles. Seawater was shown to have reduced the ORP value, which was attributed to an increase in the concentration of reducing agents such as sulphites or the reduction of dissolved oxygen concentration. The lack of redox potential value contribution from the particles themselves is thought to be due to insufficient interaction of the particles at the Pt electrode of the ORP probe.
The size of engineered nanomaterials makes many novel and innovative products, as evident by the increasing number of commercially available nanotechnology products. Thus, there is a huge concern surrounding the potential toxicity of these nanomaterials and there is a need to sufficiently test such materials. The goal here is to understand and control risk, and both toxicity testing and physicochemical characterisation should be conducted. Although our current understanding of risk associated with nanomaterials is limited, attempts have been made in order to assess this systematically. Recently, Aschberger et al.  have carried out a risk assessment based on several case studies. They have indicated the risk expected from metal and metal oxide nanomaterials, which was particularly relevant in the case of algae and Daphnia. They have attributed the risk from such materials their exposure to both the particles and corresponding dissolved ions.
There is a general consensus within the nanoecotoxicological community that physicochemical characterisation of nanomaterials in complex media is not a trivial matter, and so the reliability of such measurements is vital if we are to understand and control the risk imposed by nanomaterials [2, 3]. In recent years, the OECD initiative has adopted a holistic approach to this problem and that physicochemical characterisation should be carried out with as many parameters as possible, to include redox potential. The PROSPEcT (Ecotoxicology Test Protocols for Representative Nanomaterials in Support of the OECD Sponsorship Programme) project is the UK’s contribution to this OECD initiative, and the UK is responsible for two types of nanomaterials: cerium oxide (CeO2) and zinc oxide (ZnO) . Out of all seventeen OECD parameters identified, redox potential is the most ambiguous in its definition, what this parameter means and how it is measured, in a nanoecotoxicological context.
Integral to any ecotoxicological investigation is the ability to measure the redox conditions of a given system as indicated by the redox potential. In nature, redox reactions are an important part of phenomena such as mineral weathering, bacterial respiration, and degradation of pollutants . In soil chemistry, for example, the redox potential value can estimate whether the soil is aerobic or anaerobic, and whether chemical compounds such as Fe oxides or nitrate have been chemically reduced or are present in their oxidised form. In natural waters, redox reactions include the oxidation of organic matter and various reduction reactions such as the reduction of oxygen to water, nitrate to elementary nitrogen dioxide, iron (III) to Fe (II), sulphate to sulphide, and carbon dioxide to methane . In terms of nanomaterial toxicity, redox potential is a parameter that has been associated with inducing oxidative stress. Recently, Burello and Worth , in their prediction of a given nanomaterial to induce oxidative stress, have developed a theoretical framework that combines measurements of nanoparticle particle size and redox potential. The need to accurately measure redox potential is evident, in particular we need to understand the extent that nanomaterials can influence the natural redox phenomena should these materials be released into the environment.
Redox potential is a measure of a system’s affinity for electrons, and the measurement of redox potential will only have meaning when there are reduced and oxidised species, called the redox couple, in the liquid media. The redox couple undergoes a redox reaction, in which the reduction (gain of electrons) of one redox species is accompanied by the oxidation (loss of electrons) of another . The movement of electrons, governed by kinetics (e.g., transport limitations of the redox species to the electrode), creates an electric potential. The potential measured is determined by the ratio of activities of oxidised and reduced species, as defined by the Nernst equation; this is a thermodynamic property . The redox potential can be directly measured using a potentiometer (high impedance voltmeter) with an oxidation-reduction potential (ORP) electrode . This is the recommended technique under the current OECD guidelines for the testing of nanomaterials (NMs) ; essentially it is a measurement of potential difference (in mV) across a two-electrode system, that is, an inert platinum (Pt) electrode and silver-silver chloride (Ag/AgCl) reference electrode. Rogers et al. have recently adopted this approach to measure the redox potential of nanomaterial dispersions, that is, cerium oxide dispersed in synthetic freshwater algal medium .
There are theoretical and practical difficulties associated with the measurement of redox potential and these, although not well recognised, have already been discussed for many years . Firstly, redox potential is based on the concepts of equilibrium thermodynamics, and as such it can only be adequately measured at equilibrium. A reliable redox potential measurement requires that equilibrium be established not only at the electrode, but also among the various redox couples in solution. Many redox reactions are slow and often are at nonequilibrium conditions. Secondly, most redox potential measurements represent mixed potentials, and certain redox species may not contribute significantly towards the redox potential value; that is, not all will react sufficiently fast enough at the electrode and therefore will not contribute towards stable and reliable redox potential measurements . If the particles themselves act as a redox species, then there are various factors that may prevent them contributing towards the final ORP value, including sedimentation events, diffusion limitations, and the barrier of electron exchange at the Pt electrode. If this is true, then redox potentials of nanomaterial dispersions are likely to be solely dominated by dissolved redox species in the media, rather than contributions arising from the particles themselves. In this study, we aim to investigate if this is the case. Although the ORP probe has been conveniently used in the past by scientists to directly measure redox potential, it is essential that the reliability of such data should be questioned when measuring nanomaterial dispersions. There is the risk that researchers may treat such a tool as a black box and thus may not be fully aware of the inherent limitations in the use of such a tool in these measurements.
In this study, the ORP values of six nanomaterial (either ZnO or CeO2 dispersed in one of the four liquid media) dispersions will be measured using an ORP probe. Dispersions will be carried out according to the dispersion protocol as recommended under PROSPEcT. The ORP values of the nanomaterial dispersions will be compared relative to each other and to the corresponding media blank, to identify if there is any evidence of redox contributions as a result of the particles themselves. If there are redox contributions from the particles themselves, then this is likely to happen when dispersions are stable, as this would allow sufficient time for the particles to interact with the Pt electrode. Consequently as part of the investigation, the properties associated with the different nanomaterials will be characterised, parameters of interest to include particle size, zetapotential, and dispersion stability (as reported by the so-called half life values). Zeta-potential is a well-known parameter that characterizes the electric properties of solid surface in contact with liquid and is a way to probe surface charge. The magnitude of this value is related to dispersion stability, that is, the higher the value, the better the dispersion stability . The concept of “half-life” has been put forward in the OECD guidelines as the measurand to indicate dispersion stability through time that is, the larger the half-life value; the longer it takes for the concentration to reduce by half and thus the more stable the dispersion. Lastly, the corresponding scanning electron microscopy (SEM) data, that is, the primary particle size (mean Feret diameter) and the corresponding standard deviation, will also be reported.
2. Experimental Section
All experiments were performed in a temperature-controlled laboratory, and for the redox potential measurements the temperature of the dispersions were monitored using a temperature probe (reported value of ~20°C) to ensure that any change in the readings was not attributed to temperature changes in the dispersions.
The NMs supplied from the PROSPEcT programme were of two types, either CeO2 or ZnO, and are as follows:
(a) Nanograin CeO2 (from Umicore Belgium),
(b) Nanosun ZnO (from Micronisers, Australia),
(c) Micron ZnO (from Sigma Aldrich, UK),
(d) Z-COTE ZnO (from BASF, Germany),
(e) Micron CeO2 (from Sigma Aldrich, UK),
(f) Ceria dry CeO2 (from Antaria, Australia).
The particles were used as received and did not contain any added surface stabilisers.
DI water (resistivity of 18 Mohm) from a Millipore, MilliQ system was used to prepare all aqueous solutions and suspensions.
For the purpose of zetapotential measurements, DI water with 5 mM sodium chloride (Sigma Aldrich, UK) was employed in addition to deionised water; the NaCl here served as background electrolyte for the measurement of zetapotential. The “recipes” (chemical compositions) used for making up the ecotox media were obtained from the University of Exeter, one of our collaborators in the PROSPEcT project.
Three types of ecotox relevant media were prepared accordingly and for long-term storage, the ecotox solutions were autoclaved and kept refrigerated until needed.
(a) Seawater, in which 25 g per L of Tropic Marine Sea Salt (Tropical and Marine Limited) was made up, resulting in pH ~8.8.
(b) Daphnia freshwater media. This was prepared by firstly dissolving appropriate salts (196 mg CaCl2·2H2O, 82 mg MgSO4·7H2O, 65 mg NaHCO3, 0.002 mg Na2SeO3, as obtained by appropriate dilutions of a 2 mg/mL stock solution) in 1 L of DI water. Upon continued stirring, further DI water was added so that conductivity of the solution was between ~360 and 480 μS/cm. End volume ~1–1.5 L. Final pH ~7.9.
(c) Fish freshwater media. This was prepared in three separate steps. First, salts (11.76 g CaCl2·2H2O, 4.93 g MgSO4·7H2O, 2.59 g NaHCO3, 0.23 g KCl) were dissolved separately in 1 L of DI water to make four separate stock solutions. Second, 25 mL of each salt stock solution was aliquot into a clean bottle and diluted in DI water (made up to 1 L volume). Third, 200 mL of the stock solution from step 2 was aliquoted and further diluted with DI water (made up to 1 L volume). Final pH ~7.3.
Nanomaterials were dispersed using the protocol as previously reported (Tantra, Jing, Gohil 2010) (http://www.nanotechia-prospect.org/publications/basic). Briefly, this involved weighing the nanoparticle powder into small, clean vials using an analytical mass balance. Dispersion was carried out by adding the appropriate liquid media (fish, daphnia, seawater, or DI water) dropwise (5 drops from a Pasteur pipette) and mixing using a spatula so as to produce a thick paste before adding 15 mL of liquid media and stirring gently, using the same spatula. The formation of a thick paste as a first step was necessary to allow the efficient displacement of powder-air interface with the powder-liquid interface. The subsequent deagglomeration step was carried out using an ultrasonic probe (130 Watt Ultrasonic Processors); this was done by inserting the ultrasonic probe tip (6 mm Ti) half way down the 15 mL volume of dispersed nanoparticles, and sonication was carried out with 90% amplitude for 20 s. After sonication, the nanoparticle suspension was diluted using the appropriate liquid media, in order to make up to 1 L total volume. A glass rod was used to gently mix the final dispersion, to ensure homogeneity. The dispersions (in the four different media) were stored in separate precleaned 1 L media bottles and left undisturbed. Dispersion concentrations were 50 mg/L. Analyses of redox potentials, half-lives, and zetapotentials were conducted on the day immediately after the dispersions were made.
2.2. High-Resolution SEM
SEM images were obtained using a Supra 40 field emission scanning electron microscope from Carl Zeiss (Welwyn Garden City, Hertfordshire, UK), in which the optimal spatial resolution of the microscope is a few nanometres. In-lens detector images were acquired at an accelerating voltage of 15 kV, a working distance of ≈3 mm, and a tilt angle 0°. The SEM was calibrated using a SIRA grid calibration set (SIRA, Chislehurst, Kent, UK). These are metal replicas of cross-ruled gratings of area 60 mm2 with 19.7 lines/mm for low magnification and 2160 lines/mm for high magnification calibrations, accurate to 0.2%. For analysis of the “as received” nanoparticle powders, a sample of each powder was sprinkled over a SEM carbon adhesive disc; one side of the carbon disc was placed securely on a metal stub, whilst the other side was exposed to the nanoparticle powder. Excess powder was removed by gently tapping the stub on its side until a light coating of powder on the surface became apparent. An adequate magnification was chosen for image acquisition for example, for the estimation of primary particle mean diameter, the shape and limits of the primary particles should become apparent. SEM micrographs were analysed by manually tracing contours of primary particles onto a transparency sheet. The transparency sheet was scanned for further image analysis using Image J software, which automatically calculated particle diameter dimensions.
2.3. Redox Potential Measurements
Redox potentials were measured using an ORP Oakton Waterproof ORP Testr, purchased from Cole-Parmer, UK. This, in effect, measures the potential difference across two electrodes (a Pt electrode against a double junction Ag/AgCl reference electrode). The ORP instrument manufacturer has specified a resolution of ±1 mV, with an accuracy of ±2 mV.
The electrode was used in accordance with the manufacturer’s instructions. Prior to use, the electrode was preconditioned in clean tap water for 30 minutes before a final rinse with distilled water. When making measurements, the electrode was carefully placed in a vial containing the nanomaterial dispersion sample; there must be sufficient liquid sample to cover the sensing element. The electrode was carefully stirred a little and then placed in a fixed position, slightly above the bottom of the container. The signal output was allowed to settle for 5 minutes before a reading, the “field potential,” was noted. At this point, the signal was stable and there was no further change observed within the next few minutes. After measurement, the electrode was cleaned with tap water and rinsed with distilled water, after which further measurements could be made. When not in use, the electrode was stored in Oakton electrode storage solution.
The redox potential ORP electrode was calibrated against YSI Zobell ORP Calibration Solution (purchased from Cole-Palmer). This reagent was made available in dry form and was reconstituted with 125 mL of DI water prior to use, after which the solution has ~6-month expiry date. This standard solution was used to verify the performance of the electrode at the beginning and end of the study. For Ag/AgCl reference, the redox potential value for Zobell solution was quoted to be 231 ± 10 mV (depending on temperature); at ~20°C, this value was ~237 mV.
Redox potential measurements were carried out on freshly dispersed nanomaterial in the four chosen media, as detailed above. All field potential values recorded were subjected to an additive correction factor of +206 mV. This was necessary so that the final value was reported as if the reference electrode was a standard hydrogen reference electrode (SHE) instead of the Ag/AgCl, as previously documented . The conversion from Ag/AgCl to SHE is typically on the order of 200 to 220 mV, and voltage correction is temperature dependent and also varies slightly with the concentration of KCl (~3.5 M) in the electrode filling solution.
2.4. Measurement of Half-Lives through Turbidity Measurements
Turbidity was measured using an HF Scientific-Micro100 RI turbidity meter (Cole-Palmer, UK); this meter has an infrared light source that meets the international standard ISO 7027 for turbidity measurements. The meter was calibrated on standards, which are based on AMCO-AEPA-1 microspheres; these standards are traceable to standard formazin suspension. Standard values of 1000, 10 and 0.02 NTU were used to calibrate the meter. Prior to use, the meter was allowed to warm up for 30 minutes. Sample cuvettes (HF Scientific (USA)) were used to hold the samples. Note that glass thickness may vary from cuvette to cuvette and within the same cuvette. Hence, individual vials were indexed; indexing of the cuvette entails finding the point of the cuvette that light passes through that gives the lowest reading and, once indexed, the holder can be marked accordingly. Prior to their use, cuvettes were cleaned, in accordance with manufacturer’s instructions. This involved washing the interior and exterior of the cuvette with a detergent (2% Hellmanex in DI water); it was then rinsed several times in distilled water before finally rinsing in DI water. The cuvette was further rinsed with the sample two times before filling (30 mL) and analysed. The cuvette was placed into the meter and signal allowed to settle before taking readings. Turbidity readings were taken at regular time intervals. When not in use, the vials (containing the dispersions) were stored in the dark.
2.5. Zetapotential Measurements
Electrophoretic measurements were obtained using a Zetasizer Nano ZS (Malvern Instruments, UK) equipped with a 633 nm laser. The reference standard (DTS1230, zetapotential standard from Malvern) was used to qualify the performance of the instrument. Sample preparation involved filling a disposable capillary cell (DTS1060, Malvern). Prior to their use, these cells were thoroughly cleaned with ethanol and deionised water, as recommended by the instrument vendor. For analysis, the individual cell was filled with the appropriate sample and flushed before refilling; measurement was carried out on the second filling. Malvern Instrument’s Dispersion Technology software (Version 4.0) was used for data analysis, and zetapotential values were estimated from the measured electrophoretic mobility data using the Smoluchowski equation, as documented in a previous publication .
3. Results and Discussion
Results show that ORP values are dominated by the type of liquid media used for dispersions. Nanomaterial dispersions in seawater resulted in a much smaller ORP values in comparison with dispersions made in other media. In addition, this is also the case with the ORP values of the corresponding blank media, that is, blank seawater having the smallest ORP value, of 384 mV, compared to the rest of the blank liquid media (redox potential values all above 400 mV). The ORP values reported here is not specific to a single chemical species and thus represent an aggregate oxidization-reduction of all species that can react at the Pt working electrode . The fact that the type of liquid media itself seems to have some contribution to the final ORP values is not surprising, as dissolved redox species in the liquid can easily interact with the Pt electrode of the ORP probe. In fact, such ORP measurements are often employed as an accurate gauge of water quality and for the monitoring of dissolved species in the water . Seawater in particular is shown to be more reducing in nature, that is, due to higher concentration of reducing agents (such as N O2 −) in such media, if compared to the other liquid media. The presence of reducing agent has the effect of lowering the ORP value . Furthermore, we expect a much-reduced level of oxidising agent such as dissolved oxygen in such a high saline solution, as the more saline the water can be the less oxygen the water can hold. If there is a reduction in oxidising agents, then this also has an effect of lowering the ORP value .
The ORP readings reported here were taken three times with very little variation among the replicates, that is, not more than ±2 mV. However, the second and third replicates were acquired soon after acquiring the 1st replicate, by taking the ORP probe out of the dispersion and reimmersing it back into the dispersion. The variations in the replicates here thus represent variations of the instrument’s accuracy; they will not represent any variations that might be due to other factors such as differences in dispersion quality. Table 1 also shows the change in ORP values (values reported in brackets) upon addition of the nanomaterials relative to the corresponding blank media. Results show that, in most cases, there is a change of less than ~10 mV associated upon addition of the nanomaterials. There are only three cases in which the ORP value change is greater than 15 mV: Nanograin CeO2 in fish medium, Micron CeO2 in DI water, and Ceria dry CeO2 in fish media. Currently, we offer no explanation as to why there is a much larger change in ORP values in these three cases, apart from potential variations in dispersion quality, for example, due to potential redox contaminants associated with the different samples received. In addition, no real differentiation can be made between ZnO and CeO2 particles, with Z-COTE ZnO (BASF, Germany) having the same 9 mV change as the Ceria dry CeO2 (Antaria, Australia), when both are dispersed in DI water.
Results show that zetapotential values of nanomaterials when dispersed in seawater cannot be successfully measured (due to high conductivity) and thus displayed as N/A in Table 2. Such unsuccessful measurements were reported in the corresponding “quality report” at the end of the measurement. In general, results indicate high zetapotential values for nanomaterials that are dispersed either in DI water or DI water + 5 mM NaCl. Results of the DI water are similar to the corresponding DI water + NaCl case; the addition of NaCl into the DI water was carried out so as to have greater confidence in the DI water results, as the measurement of zetapotential usually involves the presence of inert background electrolyte. Overall, results show that nanomaterials are most stable when dispersed in DI water (or DI water + NaCl) and least stable when in an ecotox media. This is true apart for the case of Micron CeO2 showing that it is least stable in DI water, that is, −7 mV when compared to other ecotox media such as fish medium, that is, −22 mV. Currently, no explanation is available for this behaviour, and dispersion stability was further measured by using the concept of half-life values (as previously discussed in the introduction, the larger the half-life, the more stable the dispersion). Results in Table 3 show that, as with zetapotential measurements, results in DI water are generally more stable (with the largest associated with Z-COTE ZnO of 4038 minutes) than when in ecotox media. However, unlike the zetapotential values, Micron CeO2 also shows the same trend, that is, most stable in DI water than when in an ecotox media. The reason for this discrepancy lies in the fact that dispersion stability was measured in two different ways: through the measurement of interparticle force (zetapotential) or through analyzing the stability via sedimentation measurements (turbidity with time). The former measurement is solely governed by the electric properties of the solid surface in contact with liquid, which will subsequently contribute towards sedimentation rate; the latter measurement is not only determined by the zetapotential value but also by other factors, for example, particle size (in which the larger particles are expected to sediment at a much faster rate) . The SEM results (Table 4) show the mean (Feret) primary particle sizes and the corresponding standard deviations (to reflect on the polydispersity of the primary particle size). Results presented in Table 4 show that the mean primary particle sizes of the samples range from ~30 nm to ~890 nm, the largest being the Micron ZnO and Micron CeO2 from Sigma Aldrich. The SD values show the large degree of polydispersity associated with samples received: high polydispersity associated with Sigma Aldrich samples, less so with Nanosun ZnO, Microniser. The SEM micrographs indicated that all particles tested here were highly aggregated together into agglomerates of irregular shape.
If there is any particle contribution towards the final ORP values, then, out of the two types of nanomaterials tested, we expected CeO2 to have a bigger contribution compared to ZnO. This is on the basis that CeO2 particle can act as a redox couple of Ce(IV)/Ce(III), which is not the case for ZnO. If CeO2 particles had contributed to the final ORP value, then we expect this to occur with Nanograin CeO2 (having the smallest particle size of ~30 nm, as shown in Table 4) and when dispersed in DI water, as this resulted in a highly stable dispersion (noted by its high dispersion stability value of 2676 min and high zeta potential value of 33 mV, as shown in Tables 3 and 2, resp.). A highly stable dispersion will mean sufficient time to allow particles to diffuse to the Pt electrode, thus interacting with the Pt electrode in order to contribute towards the final ORP reading. Hence, we expected the redox potential to be affected most by the Nanograin CeO2 in DI water and clearly this was not the case. As shown in Table 2, Nanograin CeO2 in DI water only resulted in an ORP value change of 11 mV compared to a change of 21 mV when the same particles were dispersed in fish medium. Overall, results suggest that the particles have minor effects on the final ORP readings.
The study investigated the redox potential measurements, using ORP probe electrode, of different ZnO and CeO2 dispersions, in various liquid media. The variations in the ORP readings for the different dispersions could not be regarded as being highly significant and were mainly governed by the type of liquid media that the nanomaterials were dispersed in. This is not surprising, as ORP values are dominated by the amount of dissolved chemical species in the liquid media. Dispersions in seawater resulted in the lowest ORP values, suggesting that the media is reducing in nature. This was attributed to a much higher concentration of reducing agents such as sulphites or a reduction in the concentration of dissolved oxygen under a high salinity environment. The study shows that there was little contribution from the particles themselves towards the final ORP reading, with no significant differentiation between CeO2 and ZnO. As it is clear that redox potential measurements using an ORP electrode will not indicate a particle’s contribution towards the final redox potential value, the work has highlighted the need to have better tools for such measurements. There are several alternatives to using the ORP probe, including X-ray photoelectron spectroscopy (XPS) and electron paramagnetic resonance (EPR) and electron energy loss spectroscopy (EELS) . However, these technologies rely on the indirect measurement of redox potential and the accuracy of the values reported may come into question.
This work was conducted as part of PROSPEcT, which is a public-private partnership between DEFRA, EPSRC, and TSB and the Nanotechnology Industries Association (NIA Ltd.) and its members, and was administered by the DEFRA LINK Programme. The authors would like to thank Dr. Alex Shard for continuing support and Mr. Jordan Tompkins for the initial handling and distribution of the nanomaterials. R. Tantra gratefully acknowledges Professor Philip N. Bartlett from the University of Southampton and Dr. Andy Wain, whose expertise in electrochemistry has immensely helped in our understanding of the study.
Copyright © 2012 Ratna Tantra et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Ratna Tantra, Alex Cackett, Roger Peck, Dipak Gohil, and Jacqueline Snowden, “Measurement of Redox Potential in Nanoecotoxicological Investigations,” Journal of Toxicology, vol. 2012, Article ID 270651, 7 pages, 2012. doi:10.1155/2012/270651
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