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Parties: 1,
2
Original
Publication: M
edical
Colombia,
1997, 28: 123-
129 - ISSN
1657-9534,
Reprinted with
permission:
Corporacin
Editora
Medica del
Valle,Universi
ty of Valle,
Cali,
Colombia
Summary
Materials and methods
Results
Discussion
References
SUMMARYVibrio cholerae were inoculated in bottles ofglass, plastic bottles and plastic bags containingwater, were
exposed to sunlight for 6 hours and measured thetemperature and UV-A. Upon reaching 30 , 35 , 40
and 45 C were sampled to determine the level of vibrios and thermotolerant coliforms.Impacts were
determined temperature, turbidity, container and concentration of vibrios. This bacterium is able to
inactivate gradually reach 45 C and 6 hours of sun exposure, with constant temperature of 30 C it took
100 Wh/m2, and 50 C only 10 Wh/m2. Turbidity is a factor that interferes with the processof SODIS,
with 40 105 UNT Wh/m2 needed, with 23 NTU, 91 NTU Wh/m2 and with 5, 36 Wh/m2 of UV-A to achieve
100% mortality . No differences were found with the type of container used, always achieve 100%
inactivation, no differences were observed in the inactivation with different concentrations of vibrio. It
achieved high correlations between the inactivation of thermotolerant coliforms and vibrios (0.86 and
0.99).
Keywords: Vibrio cholerae. Thermotolerant coliforms.
Drinking water. Solar disinfection. Solar radiation. Lightultraviolet.
********
Cholera has become important in America, it was established since 1991 and the end of that year was
Use of solar radiation (UV-A and temperature) on theinactivation of Vibrio cholerae in water for humanconsumption. Factors affecting the efficiency of the
Submitted byYezid Solarte Object1
Object3Object2
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extended by 16 countries 1. In Colombia, has caused several epidemics in human and economic losses,
the groupmost affected human is between 15-44 years 2.
Cholera, a disease that causesdiarrhea very intense and is considered one of the most severas1 intestinal
infections, 3, is produced by Vibrio cholerae has two biotypes: the classical and El Tor. In Colombia it was
found that 99.1% of isolates belong to subtype Inaba and one to subtype Ogawa2. The El Tor biotype is
much more resistant to environmental conditions and survive more time inthe water. We have seen that
thesebacteria can survive in water between 2 and 20 days at temperatures between 20 and 30 C,
although this depends on the presence of other microorganisms and the chemical composition of water.
Almost all bacteria are sensitive to radiation and combined application ofheat due to the synergistic effects
that offer both MEASURES4. Therefore, a combined application of these two processesin water treatment,
allows a new alternative for disinfecting water, particularly in remote areas of difficult acceso4, 5.
Solar disinfection (SODIS) is based on the use of two-liter bottles or 3 liters plastic bags that are filled with
water and exposed for 6 hours direct sunlight. With this exposure time is achieved reductions between 92%
and 99% of coliform termotolerantes6, 7 which are indicators of fecal contamination. Therefore, and
considering the level of pathogenic bacteria is much lower, it could achieve a reduction to negligible levels
for the healthof man.
In the field station of the Institute for Research and Development ofWater , Basic Sanitation and
Conservation of Water Resources (CINARA), there has been evidenceoflaboratoryand field with
thermotolerant coliforms as indicators ofefficiencyin the SODIS, with determined that the death rate, solar
radiation levels necessary to inactivate germs and the critical temperature which can cause increased
mortality of bacterias7-9.
With the results, we decided to investigate some factors that interfere with the inactivation of V. cholerae
and determine the potential use of SODIS in thecontrol of cholera, as this type of technology is universally
available,cost and low maintenance, and is used everywhere, but has some limitations due to atmospheric
conditions, the turbidity of water, the colors , the wall thickness and shape of recipiente10.
This article presents the results under laboratory conditions and discusses the potential use that could be
SODIS inactivation of V. cholerae.
MATERIALS AND METHODS
The datain this document are part of a programof research (SODIS) is performed simultaneously in
several countries and is coordinated by the Swiss Federal Institute for Environmental Science and
Technology (EAWAG / SANDEC) and which participates in the evaluation CINARA both in the laboratory
and in the field. The experimental part was conducted in the CINARA Research Station during the period
August 1995 to April 1996.
Physical measurements. The turbidity was measured with the nephelometric method using a Hach 2100A
turbidimeter, whosevalue is given in nephelometric turbidity units (NTU). Suspended solids were
measured using the gravimetric method (mg / l) and glass fiber filters with a nominal pore size of 1.2
m11. The intensity of UV-A (Watt.hora/m2) was measured every 10 minutes with a radiometric sensor
104A-SD Macam type Cos, with a spectral response of 320-400 nm (ultraviolet A, UV-A), this sensor was
connected to a data logger and temperature was measured with a thermocouple type J.
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Microbiological tests. We used a strain of El Tor biotype of V. cholerae isolated in the Department of
Microbiology at the Universidad del Valle. This strain was maintained in nutrient broth at 35.5 C for 24
hours this medium was inoculated 1 ml in each bottle, before exposure to solar radiation.
Before and after sun exposure 5 tubes were inoculated for each concentration ofsample : 0.1, 1.0 and 10 ml
containing alkaline peptone broth to determine the most probable number (MPN). These tubes were
incubated for 8 hours at 35.5 C and tubes withbacterial growth, were sampled with a handle that was
inoculated in Petri dishes with TCBS agar, these boxes were incubated for 24 h at 35.5 C. Some typical
colonies for V. cholerae were identified in each box with five standard biochemical tests: TSI, LIA, urea,
motility and indol11, 12.
With the results of these biochemical tests determined the most probable number (MPN). The tests were
conducted with waters of different turbidities, and containers and different environmental conditions. The
solar radiation exposures were made between 09:00 and 17:00 h.
Impact of temperature. A glass bottle of 1.5 liter and 2 liter bottle plastic all black painted to prevent the
passage of sunlight while allowing greater accumulation of temperature. Each bottle completely filled with
water, was added 1 ml of V. cholerae which yielded a result of 11,000 MPN/100 ml.Every 15 minutes the
water temperature measured in the vessels, when it reached 35 C, 40 C, 45 C, the maximumtemperature (47 C) and at the end of the test samples were taken for sowing in tubes alkaline peptone.
We also determined the impact of two constant temperatures (with sun exposure) on the mortality of the
vibrios, were used for this quartz tubes of 30 ml and calibrated thermostat bath at 30 or 50 C.
Influence of turbidity. Three tests were performed with their respective replies, with differentqualityof
water (level of turbidity and suspended solids) and on different days, resulting in an experimental condition
variable. For tests with raw water (40 NTU) and prefiltered (23 NTU) was used four plastic bottles and four
glass bottles, while for filtered water (5 NTU) was used only one bottle of each type to make the respective
sample. All bottles were painted black on the bottom in a longitudinal plane. Samples for the first two tests
were taken at the beginning (22 C), reaching 45 C water at the end of the test and the next day once thedesired temperature was reached, pulled a bottle of each type. For the last test, the samples were taken at
the start, reaching 30 C water, 40 C, 45 C, 50 C and at the end of the experiment.
Influence of the type of container. Exposure to solar radiation was carried out with glass bottles (1.5 liters),
plastic (2 L) and plastic bags (3 liters) with the proceduredescribed above.
Influence of the concentration of V. cholerae. We used four plastic bottles (2 liters), to which were added
water and different initial concentrations of V. cholerae: 2.5 x 106, 2.5 x 105, 2.5 x 104 and 2.5 x 103
MPN/100 ml (final concentration in thevolumetotal was between 5 x 104 and 5 x 107 NMP).Temperatures
were measured every 15 minutes and samples were taken at the beginning 22 C, 30 C, 35 C, 40 C, 45
C and at the end of the test.
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RESULTS
Impact oftemperature. The initial concentration was reached, when inoculated with 1 ml of culture was
MPN/100 ml 16.000 V. cholerae (total concentration in thevolume used, ie, amount ofwaterconsumed by
apersonper day, was 320.000 NMP bottle glassand NMP 240.000 for plastic bottles).
Bothtests showed an increase in temperature slightly different between the two types of vessels due to the
different volumes used in the bottles, but the inactivation of V. cholerae was similar (Figure 1). The
temperature began to increase slowly and steadily, reaching over 50 C first in glass bottles (225 minutes)
and then in plastic bottles (240 minutes). The reduction ofbacteriawas drastic when it reached 40 C (3
log) in the water, after 45 C the bacteria level was
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Influence of turbidity. As each test was performed on different days, different turbidities were obtained,
solar radiation, maximum temperature reached and different initial inoculum of V. cholerae. Thequalityof
the water (turbidity and suspended solids) the total radiation from UV-A and the initial concentration of
V. cholerae are presented in Table 1.
Table 1: Experimental Conditions of the tests performed with Bottles and Three Types of Water.
Type of water
Raw Prefiltered Filtered
Turbidity (NTU) 40 23 5
Suspended Solids (mg / l) 75 24
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The raw water tests were positive for V. prefiltered cholerae at 45 C. Significant differences were noted in
the radiation and thetimenecessary to inactivate vibrios in these two types of water. In raw water bottles
both plastic and glass, it took 105 Wh/m2 UV-A radiation to decrease 2.6-2.9 log (from 1.500 to 4 NMP
NMP) of vibrios in 270 minutes and it took 46 prefiltered water Wh/m2 52 C and UV-A for a 3.2-3.7 log
reduction (from 11,000 to 4 NMP NMP) of vibrios in 130 minutes.
Water with low turbidity (
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OD -
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With 48 C and 84 W/m2 UV-A (305 minutes) positive tubes were not discovered for V. cholerae (
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Thermotolerant coliforms / V. cholerae glass bottles
2
2
variables80.0/99.9
99.6/99.7
91/46
105/105
270/270
310/110
0.92
0.90
Thermotolerant coliforms / V. cholerae plastic bags
2
2
variable99.99/99.9
99.99/99.3
78/78
54/54
250/250
140/140
0.99
0.99
DISCUSSION
The different tests showed that V. cholerae is sensitive to environmental conditions, this follows from the
results. The impact of just temperature increase caused drastic reductions in the vibrios (Figures 1 and
2). This same phenomenon already observed and Metcalf13 Ciochetti. On the contrary, thermotolerant
coliforms, the radiation is much more important than temperature, these bacteria resist temperature
increases to almost 50 C without causing mortalities apreciables14.
When combined temperature and solar radiation, the reduction starts at 35 C (lower turbidity) and 100
minutes when exposed containers only to temperature inactivation is initiated at 40 C and 135 minutes. In
a similar test conducted in Nigeria, it took about five hours to about 600 W/m2 UV-A for the complete
inactivation of V cholerae13, 15. MacKenzie et al.16 achieved a reduction of only 99.9% (3 log) of vibrios in
six hours of sun exposure, but this exhibition began at midday and was not determined temperature level
reached and the UV- A cumulative.
Increasing the temperature causes major changes in some physical and chemical parameters of water,
raises the rate of chemical and biochemical reactions, decreases the solubility ofgases and increases the
metabolic rate microorganismos17. These changes could be the cause of the destruction of cellular integrity,
while UV-A affects the breathing, transportthrough the membrane, nucleic acid replication and synthesis
of protenas18 and together with the dissolved O2 produces txicos19 compounds, therefore the
temperature and radiation are synergistic.
The influence of turbidity on inactivation of V. cholerae was similar to that found in the inactivation of
coliform termotolerantes8 because high levels of turbidity in the water it took higher doses of UV-A
radiation and temperature (exposure time) to completely inactivate vibrios. Odeyemi15 Alward14 and
showed that the bacteria are inactivated rapidly by solar radiation in waters with low turbidity, which in
high turbidity waters.
The comparison of results obtained with different turbidities indicated that raw water (high turbidity) took
nearly twice the radiation that the necessary pre-filtered water (turbidity average) to cause a small
reduction in the levels of vibrios. This even when the test was performed with raw water reached 50 C and
had a low initial concentration of vibrios.
Was also observed with prefiltered water was reached faster at 45 C (110 minutes vs. 140 minutes). This
suggests that high turbidity (suspended solids and) reduce theefficiencyof SODIS, so it is recommended to
reduce turbidity before the water exposed to radiation solar20, 21.
Generally, the lowerdensityof bacteria inactivation rate is higher. Odeyemi22 found high bacteria
reductions at low densities. The same author suggests that this is due to easy penetration and inactivation
caused by sunlight when the bacteria are dispersed and individually in the water, the opposite was found in
waters with high densities, because the cells can be found clustered , shielding many bacteria of UV-
A. However, in this investigation with V. cholerae inactivation seems not to be influenced by the density of
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bacteria (Figure 1), because if the UV-A does not reach the bacteria, the temperature would be responsible
for inactivating.
Another important factor is proper SODIS recipiente7 rate, 9.10. The vessel walls serve as a filter UV-A: If
the container causes high reductions is necessary to use a longer exposure time, such as plastic bottles used
in theseexperiments to reduce about 30% of the UV- A, on the contrary, plastic bags reduced about 10%. In
addition, Sommer9 showed that plastic bags are heated much more than the other vessels in the area /
water column, which happens to be much greater than with the bottles.
When the comparisons between the inactivation of thermotolerant coliforms and vibrios, found high
correlations between the inactivation of thermotolerant coliforms and vibrios (between 0.86 and 0.99). The
lowest correlations were presented with plastic bottles and plastic bags higher. The difference between
these vessels could be the level of temperature was always higher in the water in plastic bags.
This makes evident that the impact of SODIS on the vibrios is much higher than for coliforms
termotolerantes9. These results suggest theusefulness of thermotolerant coliforms comoindicators of
mortality vibrios. These results are important because the costsof the methods used for isolation and
identification of V. cholerae, with respect to those used for thermotolerant coliforms.
Preliminary tests show that there is potential for SODIS on the control of this infection. These
considerations are also based on the results obtained by other researchers in Canad14, Suiza5 and Lbano4
with inactivation of thermotolerant coliforms.
SUMMARY: Vibrio cholerae WAS Inoculata in plastic and glass bottles and plastic bags. The containers
Were Exposed to sunlight DURING six hours.When temperature raised up to 30 C, 35 C, 40 C, and 45
C, samples of water Were taken. The Impact of: a) temperature, b) turbidity, c) container and d) initial
concentration of vibrio Were determined. The inactivated bacteria Were only with temperature, When it
raised up to 45 C. Whereas, With constant temperature of 30 C it WAS NECESSARY 100 Wh/m2 of the
radiation and at 50 C, only 10 Wh/m2. The turbidity Caused Interference with SODIS process: total
inactivation WAS Achieved with Different levels: 105 Wh/m2 (40 TNU), 91 Wh/m2 (23 TNU) and 36Wh/m2 (5 TNU). The container type and initial concentration of vibrio Different did not Have Influence on
the SODIS process. Thermotolerant coliforms Among Full correlation and V. cholerae inactivation WAS
always high (0.86 and 0.99).
REFERENCES
1.Ministry ofHealth. DIGESA. Collection and systematization of environmental conditions and
activities in the control of the epidemic of cholera.Ministry of Health of Peru. Volume V, 1994, 297 pp.
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