Prins Leopold Instituut voor Tropische Geneeskunde
Institut de Médecine Tropicale Prince Léopold
Prince Leopold Institute of Tropical Medicine
Instituto de Medicina Tropical Principe Leopoldo
Nationalestraat, 155
B – 2000 Antwerpen
Stichting van Openbaar Nut 0410.057.701
POSTGRADUATE IN TROPICAL MEDICINE AND INTERNATIONAL HEALTH
MODULE 2
CLINICAL & BIOMEDICAL SCIENCES OF TROPICAL DISEASES
Practical notes
__________________________
WATER ANALYSIS
PHILIPPE GILLET, BIRGIT DE SMET, JAN JACOBS,
JANUARY 2009
Water analysis
Practical 1
What to do:
In groups of 2 persons:
-
-
Prepare the sampling material needed (1 sampling bottle per participants).
Prepare the medium needed for the shorter method based on Lauryl
Sulphate Broth, MPN, 3 x 3 tubes. (One group will prepare tubes with
single strength broth, 12 tubes per participants + reserve; the other
group will prepare tubes with double strength, 6 tubes per participants +
reserve).
Sterilise the material and the medium.
Prepare the incubators for the water analyses.
Realise the quality control of the medium.
What to remember:
-
Factors to consider for the sterilisation.
Quality controls to consider.
Use and interpretation of quality controls.
Principe of sampling for water analyses.
Practical 1I
What to do:
In groups of 2 persons:
-
Interpret the controls.
Realise the turbidity determination of 2 water samples.
Realise the determination of chlorine residual in 2 water samples.
Interpret the results.
Individually:
-
-
Realisation of the shorter method based on Lauryl Sulphate Broth, MPN,
3 x 3 tubes on 2 different water samples, Total Coliforms + Thermotolerant
Coliforms.
Interpret the results.
What to remember:
-
Principe of water analysis (turbidity, chlorine residual and microbiological
examination of water).
Factors to consider for the microbiological examination of water.
Tables of contents
Tables of contents ..........................................................................................................3
Water analysis ................................................................................................................4
MICROBIOLOGICAL EXAMINATION OF WATER ...........................................4
Indicator organisms (summary): ............................................................................4
WATER SAMPLING................................................................................................6
A Sampling from a tap or pump outlet .................................................................7
B Sampling from reservoir....................................................................................7
C Sampling from a dug well .................................................................................7
METHODS OF MICROBIOLOGICAL ANALYSIS...............................................7
“SHORTER METHOD” BASED ON LAURYL SULPHATE BROTH: ................9
TURBIDITY MEASURMENT:..............................................................................13
RESIDUAL FREE CHLORINE TEST ...................................................................14
Target values:.......................................................................................................15
ANNEX 1 : LAURYL SULFATE BROTH, MSDS ............................................16
ANNEX 2 : SODIUM THIOSULFATE PENTAHYDRATE, MSDS ...............20
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Water analysis
Water, of adequate quantity and quality, is essential for healthy life. The associations between
sanitation, water and health are well known. Many diseases are associated with contaminated
water and water shortages.
The most important factor to take into account is that, in most communities, the principal risk
to human health derives from faecal contamination. In some countries there may also be
hazards associated with specific chemical contaminants such as fluoride or arsenic, but the
levels of these substances are unlikely to change significantly with time. Thus, if a full range
of chemical analyses is undertaken on new water sources and repeated thereafter at fairly
long intervals, chemical contaminants are unlikely to present an unrecognized hazard. In
contrast, the potential for faecal contamination in untreated or inadequately treated
community supplies is always present. The minimum level of analysis should therefore
include testing for indicators of faecal pollution (thermotolerant (faecal) coliforms), turbidity,
and chlorine residual and pH (if the water is disinfected with chlorine).
MICROBIOLOGICAL EXAMINATION OF WATER
Microbiological examination offers the most sensitive test for the detection of recent and
potentially dangerous faecal pollution, thereby providing a hygienic assessment of water
quality with high sensitivity and specificity. For this reason it is important to examine a drinking
water source frequently by a simple test rather than infrequently by a more complicated test
or series of tests.
It is ideal to look for individual specific pathogen but it is not practical since they are few in
numbers than the non-pathogenic organisms and methods to detect them are costly in time
and money. Therefore indicators of human/animal pollution e.g. coliforms are used. Faecal
streptococci are regularly present in the faeces in varying numbers but their number is fewer
than Escherichia coli and they probably die and disappear at the same rate. The presence of
faecal streptococci along with coliforms in absence of Escherichia coli is also confirmatory of
faecal pollution.
Indicator organisms (summary):
Escherichia coli is a member of the family Enterobacteriaceae, and is characterized by
possession of the enzymes b-galactosidase and b-glucuronidase. It grows at 44–45°C on
complex media, ferments lactose and mannitol with the production of acid and gas, and
produces indole from tryptophan. However, some strains can grow at 37 °C but not at 44–
45°C, and some do not produce gas. E. coli does not produce oxidase or hydrolyse urea.
Complete identification of the organism is too complicated for routine use, but a number of
tests have been developed for rapid and reliable identification. Some of these methods have
been standardized at international and national levels and accepted for routine use; others
are still being developed or evaluated.
Escherichia coli is abundant in human and animal faeces; in fresh faeces it may attain
9
concentrations of 10 per gram. It is found in sewage, treated effluents, and all natural waters
and soils subject to recent faecal contamination, whether from humans, wild animals, or
agricultural activity. Recently, it has been suggested that E. coli may be present or even
multiply in tropical waters not subject to human faecal pollution. However, even in the
remotest regions, faecal contamination by wild animals, including birds, can never be
excluded. Because animals can transmit pathogens that are infective in humans, the
presence of E. coli or thermotolerant coliform bacteria must not be ignored, because the
09 01 29_PG_Bacteriological Examination of Water
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presumption remains that the water has been faecally contaminated and that treatment has
been ineffective.
Thermotolerant coliform bacteria: Thermotolerant coliform bacteria are the coliform
organisms that are able to ferment lactose at 44–45°C; the group includes the genus
Escherichia and some species of Klebsiella, Enterobacter, and Citrobacter. Thermotolerant
coliforms other than E. coli may also originate from organically enriched water such as
industrial effluents or from decaying plant materials and soils. For this reason, the term
“faecal” coliforms, although frequently employed, is not correct, and its use should be
discontinued. Regrowth of thermotolerant coliform organisms in the distribution system is
unlikely unless sufficient bacterial nutrients are present, unsuitable materials are in contact
with the treated water, the water temperature is above 13 °C, and there is no free residual
chlorine. In most circumstances, concentrations of thermotolerant coliforms are directly
related to that of E. coli. Their use in assessing water quality is therefore considered
acceptable for routine purposes, but the limitations with regard to specificity should always be
borne in mind when the data are interpreted. If high counts of thermotolerant coliforms are
found in the absence of detectable sanitary hazards, additional confirmatory tests specific for
E. coli should be carried out. National reference laboratories developing national standard
methods are advised to examine the specificity of the thermotolerant coliform test for E. coli
under local conditions. Because thermotolerant coliform organisms are readily detected, they
have an important secondary role as indicators of the efficiency of water-treatment processes
in removing faecal bacteria. They may therefore be used in assessing the degree of treatment
necessary for waters of different quality and for defining performance targets for removal of
bacteria.
Coliform organisms (total coliforms): Coliform organisms have long been recognized as a
suitable microbial indicator of drinking-water quality, largely because they are easy to detect
and enumerate in water. The term “coliform organisms” refers to Gram-negative, rod-shaped
bacteria capable of growth in the presence of bile salts or other surface-active agents with
similar growth-inhibiting properties and able to ferment lactose at 35–37°C with the production
of acid, gas, and aldehyde within 24–48 hours. They are also oxidase-negative and nonspore-forming and display b-galactosidase activity. Traditionally, coliform bacteria were
regarded as belonging to the genera Escherichia, Citrobacter, Enterobacter, and Klebsiella.
However, as defined by modern taxonomical methods, the group is heterogeneous. It
includes lactosefermenting bacteria, such as Enterobacter cloacae and Citrobacter freundii,
which can be found in both faeces and the environment (nutrient-rich waters, soil, decaying
plant material) as well as in drinking-water containing relatively high concentrations of
nutrients, as well as species that are rarely, if ever, found in faeces and may multiply in
relatively good-quality drinking-water, e.g. Serratia fonticola, Rabnella aquatilis, and
Buttiauxella agrestis. The existence both of non-faecal bacteria that fit the definitions of
coliform bacteria and of lactose-negative coliform bacteria limits the applicability of this group
as an indicator of faecal pollution. Coliform bacteria should not be detectable in treated water
supplies and, if found, suggest inadequate treatment, post treatment contamination, or
excessive nutrients. The coliform test can therefore be used as an indicator both of treatment
efficiency and of the integrity of the distribution system. Although coliform organisms may not
always be directly related to the presence of faecal contamination or pathogens in drinkingwater, the coliform test is still useful for monitoring the microbial quality of treated piped water
supplies. If there is any doubt, especially when coliform organisms are found in the absence
of thermotolerant coliforms and E. coli, identification to the species level or analyses for other
indicator organisms may be undertaken to investigate the nature of the contamination.
Sanitary inspections will also be needed.
Faecal streptococci: Faecal streptococci are those streptococci generally present in the
faeces of humans and animals. All possess the Lancefield group D antigen. Taxonomically,
they belong to the genera Enterococcus and Streptococcus. The taxonomy of Enterococci
has recently undergone important changes, and detailed knowledge of the ecology of many of
the new species is lacking; the genus Enterococcus now includes all streptococci that share
certain biochemical properties and have a wide tolerance of adverse growth conditions—E.
avium, E. casseliflavus, E. cecorum, E. durans, E. faecalis, E. faecium, E. gallinarum, E.
09 01 29_PG_Bacteriological Examination of Water
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hirae, E. malodoratus, E. mundtii, and E. solitarius. Most of these species are of faecal origin
and can generally be regarded as specific indicators of human faecal pollution for most
practical purposes. They may, however, be isolated from the faeces of animals, and certain
species and subspecies, such as E. casseliflavus, E. faecalis var. liquefaciens, E.
malodoratus, and E. solitarius, occur primarily on plant material. In the genus Streptococcus,
only S. bovis and S. equinus possess the group D antigen and therefore belong to the faecal
streptococcus group. They derive mainly from animal faeces. Faecal streptococci rarely
multiply in polluted water, and they are more persistent than E. coli and coliform bacteria.
Their primary value in water-quality examination is therefore as additional indicators of
treatment efficiency. Moreover, streptococci are highly resistant to drying and may be
valuable for routine control after new mains are laid or distribution systems are repaired, or for
detecting pollution of ground waters or surface waters by surface run-off.
WATER SAMPLING
Several type of bottle may be used for sampling, but glass bottles are best. These should
have securely fitting stoppers with non toxic liners. The bottles should hold at least 200 ml of
water and should be sterilized.
When water that contains or may contain even traces of chlorine is sampled, the chlorine
must be inactivated. If it is not, microbes may be killed during transit and an erroneous result
will be obtained. The bottles in which the samples are placed should therefore contain sodium
thiosulfate to neutralize any chlorine present. The sodium thiosulfate should be added to the
bottles before they are sterilized.
For 200 ml samples, five drops of aqueous sodium thiosulfate solution [100 g/litter (w/v)]
should be added to each clean sample bottle. The stopper is loosely adapted to the bottle
and aluminium foil cover is tied to the neck of the bottle to prevent dust from entering. The
bottle is the sterilized [in hot-air oven for 1 hour at 160°C or for 40 minutes at 170°C; or in an
autoclave at 121°C for 20 minutes.
Although recommendations vary, the time between sample collection and analysis should, in
general, not exceed 6 hours, and 24 hours is considered the absolute maximum. . It is
assumed that the samples are immediately placed in a lightproof insulated box containing
melting ice or ice-packs with water to ensure rapid cooling. If ice is not available, the
transportation time must not exceed 2 hours. It is imperative that samples are kept in the dark
and that cooling is rapid. If these conditions are not met, the samples should be discarded.
The cool box used to carry samples should be cleaned and disinfected after each use to
avoid contaminating the surfaces of the bottles and the sampler’s hands.
Sources of water to be sampled
Water sources can be divided into three basic types for the purpose of sampling.
a)
b)
c)
Water from a tap or fixed hand pump
Water from a reservoir (river, lake, tank, …)
Water from a dug well
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A Sampling from a tap or pump outlet
1.
2.
3.
4.
Remove any attachments from tap that may cause splashing.
Wipe off the dirt from outside the tap.
Turn on the tap at maximum flow rate and let the water flow for 1-2 minutes.
Sterilize it for a minute with flame using gas burner, lighter or ignited cotton
wool soaked in spirit.
5. Open the tap and allow water to flow at medium rate for 1-2 minutes.
6. Open the container for collecting the sample and fill the water by holding the
bottle under the water jet. Leave a small airspace to facilitate shaking at the
time of inoculation prior to analysis.
7. Stopper the cap and label the container.
B Sampling from reservoir
1. Submerge the bottle in the water.
2. Open the bottle inside of the water.
3. Fill it by holding it by the lower part, submerging it to a depth of about 30
centimetres, with the mouth facing slightly upwards. If there is a current, the
bottle should face the current.
4. Pull it out when the bottle is filled.
5. Discard a little water to provide airspace
6. Stopper the bottle and label it.
C Sampling from a dug well
1.
2.
3.
4.
Attach a stone of suitable size to the sampling bottle with a piece of string.
Tie a 20 meter length of clean string on the bottle and to a stick.
Open the bottle as described above and lower into the well.
Immerse the bottle completely in water without touching the sides of the well
and lower it down to the bottom of the well.
5. Pull it out when the bottle is filled.
6. Discard a little water to provide airspace.
7. Stopper and label the bottle.
METHODS OF MICROBIOLOGICAL ANALYSIS
Two methods have been developed for the detection of indicator bacteria in water:
membrane filter method and multiple tube method.
1. Membrane filtration method: In the
membrane-filtration (MF) method, a minimum
volume of 10 ml of the sample (or dilution of the
sample) is introduced aseptically into a sterile
or properly disinfected filtration assembly
containing a sterile membrane filter (nominal
pore size 0.2 or 0.45 µm). A vacuum is applied
and the sample is drawn through the
membrane filter. All indicator organisms are
retained on or within the filter, which is then
transferred to a suitable selective culture
medium in a Petri dish. Following a period of
resuscitation, during which the bacteria become acclimatized to the new conditions, the
Petri dish is transferred to an incubator at the appropriate selective temperature where it
is incubated for a suitable time to allow the replication of the indicator organisms.
09 01 29_PG_Bacteriological Examination of Water
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Visually identifiable colonies are formed and counted, and the results are expressed in
numbers of “colony forming units” (CFU) per 100 ml of original sample.
This technique is inappropriate for waters with a level of turbidity that would cause the
filter to become blocked before an adequate volume of water had passed through. When
it is necessary to process low sample volumes (less than 10 ml), an adequate volume of
sterile diluent must be used to disperse the sample before filtration and ensure that it
passes evenly across the entire surface of the membrane filter. Membrane filters may be
expensive in some countries.
Sample volumes for different water types are:
Where the quality of the water is totally unknown, it may be advisable to test two or more
volumes in order to ensure that the number of colonies on the membrane is in the
optimal range for counting (20–80 colonies per membrane).
A full description of this method (also with video) is available on internet:
http://www.rcpeh.com/index.php?option=com_content&task=view&id=118&Itemid=152
2. Multiple-tube method: In the multiple-tube method (MT) different amounts of water
to be tested are added to tubes containing a suitable culture medium. The bacteria
present in the water reproduce and produce acid with or without gas. From the number
of tubes inoculated and the number with a positive reaction, the most probable number
(MPN) of bacteria present in the original water sample can be determined statistically.
The multiple-tube method is applicable to all kinds of water: it can be used with clear,
coloured, or turbid water containing sewage or sewage sludge, or mud and soil particles,
provided that the bacteria are homogeneously distributed in the prepared test samples.
Example of culture media for multiple tube method and MPM
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“SHORTER METHOD” BASED ON LAURYL SULPHATE BROTH:
(e.g. FLUKA N°: 17349)
Double strength broth: Dissolve 71.2 g of lauryl sulphate broth in 1 litre distilled water.
After solubilisation, dispense 10 ml into each test tube containing inverted Durhan-tubes.
Sterilise by autoclaving at 121°C for 15 minutes. Cool down slowly to prevent bubbles in
Durhan-tubes.
Single strength broth: Dissolve 35.6 g of lauryl sulphate broth in 1 litre distilled water.
After solubilisation, dispense 10 ml into each test tube containing inverted Durhan-tubes.
Sterilise by autoclaving at 121°C for 15 minutes. Cool down slowly to prevent bubbles in
Durhan-tubes.
Total Coliforms:
Arrange three rows of 3 (5) tubes each in a test-tube rack. The tubes in the first row hold
10 ml of double strength lauryl sulphate broth, while the tubes in the second and the third
rows contain 10 ml of single strength lauryl sulphate broth.
With a sterile pipette (syringe) add 10 ml of the sample to each of the 3 (5) tubes of the
first row.
With a sterile pipette (syringe) add 1 ml of the sample to each of the 3 (5) tubes of the
second row.
With a sterile pipette (syringe) add 0.1 ml of the sample to each of the 3 (5) tubes of the
third row. Add also in each tube 0.9 ml of sterile distilled water.
1. Incubate the tubes at 37°C for 18-24 hours.
2. Observe change in turbidity and appearance of gas in Durham tubes in bottles.
3. The media receiving one or more of the indicator bacteria show growth (turbidity)
and a gas production which is absent in those receiving an inoculum of water
without indicator bacteria. Presence of grow and gas indicates positive reaction
whereas absence of either or both these features denotes a negative reaction.
4. The presumptive positives are read and remaining negative bottles are reincubated for another 24 hours. Any further positives are added to the previous
figures. The probable numbers of coliforms are read from the probability tables of
McCrady (Tables in annex).
5. From the number and distribution of positive and negative reactions, count of the
most probable number (MPN) of indicator organisms in the sample may be
estimated by reference to statistical tables. The test gives presumptive coliforms
count as the reaction observed may occasionally be due to the presence of some
organisms other than coliforms.
For every tube showing fermentation (primary fermentation, presumptive coliforms), you
may inoculate one new tube of Lauryl sulphate broth, from the tube showing primary
fermentation, and incubated this tube at 44°C respectively. If there is fermentation in the
tube incubated at 44°C after 8 to 24 hours, perform indole test by adding Kovac’s
reagent. A positive indole test in a broth tube showing gas production at 44°C indicates
the presence of E. coli.
From the number and distribution of positive and negative reactions at 44°C, count of the
most probable number (MPN) of indicator organisms in the sample may be estimated by
reference to statistical tables. The test gives E. coli count
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Thermotolerant coliforms + indole (E. coli):
Arrange three rows of 3 (5) tubes each in a test-tube rack. The tubes in the first row hold
10 ml of double strength lauryl sulphate broth, while the tubes in the second and the third
rows contain 10 ml of single strength lauryl sulphate broth.
With a sterile pipette (syringe) add 10 ml of the sample to each of the 3 (5) tubes of the
first row.
With a sterile pipette (syringe) add 1 ml of the sample to each of the 3 (5) tubes of the
second row.
With a sterile pipette (syringe) add 0.1 ml of the sample to each of the 3 (5) tubes of the
third row. Add also in each tube 0.9 ml of sterile distilled water.
1. Incubate the tubes at 44°C for 18-24 hours.
2. Observe change in turbidity and appearance of gas in Durham tubes in bottles.
3. The media receiving one or more of the indicator bacteria show growth (turbidity)
and a gas production which is absent in those receiving an inoculum of water
without indicator bacteria. Presence of grow and gas indicates positive reaction
whereas absence of either or both these features denotes a negative reaction.
4. The presumptive positives are read, (perform indole test by adding 0.5 ml of
Kovac’s reagent: a positive indole test in a broth tube showing gas production at
44°C indicates the presence of E. coli) and remaining negative bottles are reincubated for another 24 hours. Any further positives are tested with Kovacs and
added to the previous figures. The probable numbers of E coli are read from the
probability tables of McCrady (Tables in annex).
5. From the number and distribution of positive and negative reactions, count of the
most probable number (MPN) of indicator organisms in the sample may be
estimated by reference to statistical tables. The test gives E. coli count.
Turbidity of the medium,
accompanied by formation of
gas (trapped in the Durhamtubes) within 48 hours is a
positive presumptive for coliform
bacteria.
09 01 29_PG_Bacteriological Examination of Water
Kovac’s
test:
The
test
organism is cultured in a
medium
which
contains
tryptophane.
The breaking
down of the tryp-tophane with
the releasing of indole is
detected by the Kovac’s reagent.
This reacts with the indole to
produce
a
red
coloured
compound.
Thermotolerant bacteria with
indole production is a positive
presumptive for E. coli.
10 / 24
Kovac's reagent: (this reagent is available commercially (e.g. VWR N°1.09293.0100).
Amyl or isoamyl alcohol C5H12O
150 ml
4-Dimethylaminobenzaldehyde C9H11NO 10 g
Concentrated HCl
50 ml
Dissolve the 4-dimethylaminobenzaldehyde, C9H11NO, in 75 ml of isoamyl alcohol, C5H12O, and heat in
a water bath at 60 °C for 5 min. Then add 25 ml of concentrated hydrochloric acid. The reagent will be
ready for use after about 6 h to 7 h (indicated by a light yellow color).
If a brown color results, the reagent should not be used. Kovac's reagent is stable at 4°C for 1 year.
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TURBIDITY MEASURMENT:
High levels of turbidity can protect microorganisms from the effects of disinfection, stimulate
the growth of bacteria, and exert a significant chlorine demand. Where disinfection is
practiced, the turbidity must always be low, e.g. below 5 NTU (Nephelometric Turbidity units,
or JTU for JacksonTurbidity Units), and ideally below 1 NTU for effective disinfection.
Measurement of turbidities lower than 5 NTU will generally require electronic meters.
However, turbidities of 5 NTU upwards can be measured by simple extinction methods, which
are far cheaper and require no consumables. In the monitoring of small community supplies in
developing countries, such methods may be preferable. The sequence of steps involved in
turbidity determination by an extinction method is shown below.
Turbidity may change during sample transit and storage, and should therefore be measured
on site at the time of sampling.
Add water slowly to the turbidity tube, taking care not to form bubbles. Fill until the mark at the
bottom of the tube just disappears.
Read the turbidity from the scale marked on the side of the tube. The value is that
corresponding to the line nearest to the level of the water in the tube. The scale is not linear,
and extrapolation of values between the lines is therefore not recommended.
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RESIDUAL FREE CHLORINE TEST
The method recommended for the determination of chlorine residual in drinking water
employs N,N-diethyl-p-phenylenediamine, more commonly referred to as DPD. Methods
employing orthotolidine and starch–potassium iodide were formerly also recommended.
The first of these reagents is a recognized carcinogen and the method is not reliable. The
method based on the use of starch–potassiumiodide is not specific for free chlorine, but
measures directly the total of free and combined chlorine; it is not recommended except
in countries where DPD cannot be obtained or prepared. In this Annex, therefore, only
the DPD method is considered.
In the laboratory, colorimetry or spectrophotometry may both be used for the
determination of chlorine by means of DPD. However, it is common practice and highly
recommended for field measurements using simple color match comparators to be done
on site. The color is generated following the addition of DPD to the water sample and is
matched against standard colored discs or tubes. The method can be used by staff
without extensive specialized training. The reagent may be solid (e.g. individually
wrapped tablets) or in the form of a solution; the former is more stable. If the solution is
used, it should be stored in a brown bottle and discarded as soon as it starts to become
discolored.
It is important to measure pH at the same time as chlorine residual since the efficacy of
disinfection with chlorine is highly pH-dependent: where the pH exceeds 8.0, disinfection is less
effective. To check that the pH is in the optimal range for disinfection with chlorine (less than
8.0), simple tests may be conducted in the field using comparators such as that used for chlorine
residual. With some chlorine comparators, it is possible to measure pH and chlorine residual
simultaneously. Alternatively, portable pH electrodes and meters are available. If these are used in
the laboratory, they must be calibrated against fresh pH standards at least daily; for field use, they
should be calibrated immediately before each test. Results may be inaccurate if the water has a
low buffering capacity.
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Target values:
Class
Grade
Presumptive total coliform
count (per 100 ml)
E. coli count (per 100 ml)
I
Excellent
0
0
II
Satisfactory
1-3
0
III
Suspicious
4-10
0
IV
Unsatisfactory
>10
0,1 or more
International Organization for Standardization (ISO) standards for detection and
enumeration of faecal indicator bacteria in water ISO standard Title (water quality)
The objective of zero E. coli per 100 ml of water is the goal for all water supplies and should
be the target even in emergencies; however, it may be difficult to achieve in the immediate
post-disaster period. This highlights the need for appropriate disinfection. An indication of a
certain level of faecal indicator bacteria alone is not a reliable guide to microbial water safety.
Some faecal pathogens, including many viruses and protozoan cysts and oocysts, may be
more resistant to treatment (e.g., by chlorine) than common faecal indicator bacteria. More
generally, if a sanitary survey suggests the risk of faecal contamination, then even a very low
level of faecal contamination may be considered to present a risk, especially during an
outbreak of a potentially waterborne disease, such as cholera.
Drinking-water should be disinfected in emergency situations, and an adequate disinfectant
residual (e.g., chlorine) should be maintained in the system. Turbid water should be clarified
wherever possible to enable disinfection to be effective. Minimum target concentrations for
chlorine at point of delivery are 0.2 mg/liter in normal circumstances and 0.5 mg/liter in highrisk circumstances. It is necessary to know the pH of water, because more alkaline water
requires a longer contact time or a higher free residual chlorine level at the end of the contact
time for adequate disinfection (0.4–0.5 mg/liter at pH 6–8, rising to 0.6 mg/liter at pH 8–9;
chlorination may be ineffective above pH 9).
Typical sample volumes and numbers of tubes for the multiple-tube method
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How Chlorine Kills Pathogens
How does chlorine carry out its well-known role of making
water safe? Upon adding chlorine to water, two chemical
species, known together as “free chlorine,” are formed.
These species, hypochlorous acid (HOCl, electrically
neutral) and hypochlorite ion (OCl-, electrically negative),
behave very differently. Hypochlorous acid is not only
more reactive than the hypochlorite ion, but is also a
stronger disinfectant and oxidant.
The ratio of hypochlorous acid to hypochlorite ion in water
is determined by the pH. At low pH (higher acidity),
hypochlorous acid dominates while at high pH hypochlorite ion dominates. Thus, the speed
and efficacy of chlorine disinfection against pathogens may be affected by the pH of the
water being treated. Fortunately, bacteria and viruses are relatively easy targets of
chlorination over a wide range of pH. However, treatment operators of surface water
systems treating raw water contaminated by the parasitic protozoan Giardia may take
advantage of the pH-hypochlorous acid relationship and adjust the pH to be effective against
Giardia, which is much more resistant to chlorination than either viruses or bacteria.
Another reason for maintaining a predominance of hypochlorous acid during treatment has
to do with the fact that pathogen surfaces carry a natural negative electrical charge. These
surfaces are more readily penetrated by the uncharged, electrically neutral hypochlorous
acid than the negatively charged hypochlorite ion. Moving through slime coatings, cell walls
and resistant shells of waterborne microorganisms, hypochlorous acid effectively destroys
these pathogens. Water is made microbiologically safe as pathogens either die or are
rendered incapable of reproducing.
A typical bacterium has a negatively charged slime coating on its exterior cell wall, which is
effectively penetrated by electrically neutral hypochlorous acid, favored by lower pH’s.
(Reprinted from The Chlorination/Chloramination Handbook by permission. Copyright ©
1996, American Water Works Association.)
Source: Connell, 1996.
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ANNEX 1: LAURYL SULFATE BROTH, MSDS
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ANNEX 2: SODIUM THIOSULFATE PENTAHYDRATE, MSDS
Fisher Products, Material Safety Data Sheet
ACC# 21715 Section 1 - Chemical Product and Company
Identification
MSDS Name: Sodium Thiosulfate Pentahydrate
Catalog Numbers: S78930, NC9503498, NC9629890, NC9671016, NC9979554,
S445-10, S445-3, S445-50, S445-500, S474-12, S474-3, S474-500,
XXS445100KG, XXS47450KG
Synonyms: Sodium Hyposulfite; Disodium Salt Pentahydrate; Disodium
Thiosulfate Pentahydrate.
Company Identification:
Fisher Scientific
1 Reagent Lane
Fair Lawn, NJ 07410
For information, call: 201-796-7100
Emergency Number: 201-796-7100
For CHEMTREC assistance, call: 800-424-9300
For International CHEMTREC assistance, call: 703-527-3887
Section 2 - Composition, Information on Ingredients
CAS#Chemical NamePercentEINECS/ELINCS
10102-17-7Sodium Thiosulfate, Pentahydrate100 unlisted
Hazard Symbols: None listed.
Risk Phrases: None listed.
Section 3 - Hazards Identification
EMERGENCY OVERVIEW
Appearance: colourless to white solid. Caution! May cause eye and
skin
irritation. Hygroscopic (absorbs moisture from the air). The
toxicological properties of this material have not been fully
investigated. May cause respiratory and digestive tract irritation.
Target Organs: No data found.
Potential Health Effects
Eye: May cause eye irritation.
Skin: Prolonged and/or repeated contact may cause irritation and/or
dermatitis.
Ingestion: Ingestion of large amounts may cause gastrointestinal
irritation. The toxicological properties of this substance have not
been fully investigated.
Inhalation: May cause respiratory tract irritation. Low hazard for
usual industrial handling. The toxicological properties of this
substance have not been fully investigated.
Chronic: No information found.
Section 4 - First Aid Measures
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Eyes: Flush eyes with plenty of water for at least 15 minutes,
occasionally lifting the upper and lower eyelids. Get medical aid.
Skin: Flush skin with plenty of water for at least 15 minutes while
removing contaminated clothing and shoes. Get medical aid if
irritation develops or persists. Wash clothing before reuse.
Ingestion: Never give anything by mouth to an unconscious person. Get
medical aid. Do NOT induce vomiting. If conscious and alert, rinse
mouth and drink 2-4 cupfuls of milk or water.
Inhalation: Remove from exposure and move to fresh air immediately.
If not breathing, give artificial respiration. If breathing is
difficult, give oxygen.
Get medical aid.
Notes to Physician: Treat symptomatically and supportively.
Section 5 - Fire Fighting Measures
General Information: As in any fire, wear a self-contained breathing
apparatus in pressure-demand, MSHA/NIOSH (approved or equivalent),
and full protective gear. During a fire, irritating and highly toxic
gases may be generated by thermal decomposition or combustion. Wear
appropriate protective clothing to prevent contact with skin and
eyes. Wear a self-contained breathing apparatus (SCBA) to prevent
contact with thermal decomposition products.
Extinguishing Media: Use agent most appropriate to extinguish fire.
Use water spray, dry chemical, carbon dioxide, or appropriate foam.
Flash Point: Not applicable.
Autoignition Temperature: Not applicable.
Explosion Limits, Lower: Not available. Upper: Not available.
NFPA Rating: (estimated) Health: 1; Flammability: 0; Instability: 0
Section 6 - Accidental Release Measures
General Information: Use proper personal protective equipment as
indicated in Section 7.
Spills/Leaks: Vacuum or sweep up material and place into a suitable
disposal container. Clean up spills immediately, observing
precautions in the Protective Equipment section. Avoid generating
dusty conditions. Provide ventilation.
Section 7 - Handling and Storage
Handling: Wash thoroughly after handling. Use with adequate
ventilation.
Minimize dust generation and accumulation. Avoid prolonged or
repeated contact with skin. Avoid contact with eyes, skin, and
clothing. Keep container tightly closed. Avoid ingestion and
inhalation.
Storage: Store in a tightly closed container. Keep from contact with
oxidizing materials. Store in a cool, dry, well-ventilated area away
from incompatible substances. Keep away from strong acids. Store
protected from moisture.
Section 8 - Exposure Controls, Personal Protection
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Engineering Controls: Facilities storing or utilizing this material
should be equipped with an eyewash facility and a safety shower. Use
adequate ventilation to keep airborne concentrations low.
Exposure Limits Chemical NameACGIHNIOSHOSHA - Final PELs
Sodium Thiosulfate, Pentahydratenone listednone listednone listed
OSHA Vacated PELs: Sodium Thiosulfate, Pentahydrate: No OSHA Vacated
PELs are listed for this chemical.
Personal Protective Equipment
Eyes: Wear appropriate protective eyeglasses or chemical safety
goggles as described by OSHA's eye and face protection regulations in
29 CFR 1910.133 or European Standard EN166.
Skin: Wear appropriate protective gloves to prevent skin exposure.
Clothing: Wear appropriate protective clothing to prevent skin
exposure.
Respirators: A respiratory protection program that meets OSHA's 29
CFR 1910.134 and ANSI Z88.2 requirements or European Standard EN 149
must be followed whenever workplace conditions warrant a respirator's
use.
Section 9 - Physical and Chemical Properties
Physical State: Solid
Appearance: colourless to white
Odour: odourless
pH: Not available.
Vapour Pressure: Not available.
Vapour Density: Not available.
Evaporation Rate: Not available.
Viscosity: Not available.
Boiling Point: Not available.
Freezing/Melting Point: 48.5 deg C
Decomposition Temperature: > 45 deg C
Solubility: 680 G/L WATER (20óC)
Specific Gravity/Density:1.7290g/cm3
Molecular Formula: Na2O3S2.5H2O
Molecular Weight: 248.18
Section 10 - Stability and Reactivity
Chemical Stability: Stable.
Conditions to Avoid: Incompatible materials, acids, strong oxidants,
exposure to moist air or water.
Incompatibilities with Other Materials: Moisture, strong acids,
strong bases, strong oxidizing agents.
Hazardous Decomposition Products: Irritating and toxic fumes and
gases, hydrogen sulphide, sodium oxide.
Hazardous Polymerization: Has not been reported.
Section 11 - Toxicological Information
RTECS#:
CAS# 10102-17-7: WE6660000
LD50/LC50: Not available.
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Carcinogenicity: CAS# 10102-17-7: Not listed by ACGIH, IARC, NIOSH,
NTP, or OSHA.
Epidemiology: No information available.
Teratogenicity: No information available.
Reproductive Effects: No information available.
Neurotoxicity: No information available.
Mutagenicity: No information available.
Other Studies: See actual entry in RTECS for complete information.
Section 12 - Ecological Information
No information available.
Section 13 - Disposal Considerations
Chemical waste generators must determine whether a discarded chemical
is classified as a hazardous waste. US EPA guidelines for the
classification determination are listed in 40 CFR Parts 261.3.
Additionally, waste generators must consult state and local hazardous
waste regulations to ensure complete and accurate classification.
RCRA P-Series: None listed.
RCRA U-Series: None listed.
Section 14 - Transport Information
US DOTIATARID/ADRIMOCanada TDG
Shipping Name: No information available.
Hazard Class:
UN Number:
Packing Group:
Section 15 - Regulatory Information
US FEDERAL
TSCA
CAS# 10102-17-7 is not on the TSCA Inventory because it is a hydrate.
It is considered to be listed if the CAS number for the anhydrous
form is on the inventory (40CFR720.3(u)(2)).
Health & Safety Reporting List
None of the chemicals are on the Health & Safety Reporting List.
Chemical Test Rules
None of the chemicals in this product are under a Chemical Test Rule.
Section 12b
None of the chemicals are listed under TSCA Section 12b.
TSCA Significant New Use Rule
None of the chemicals in this material have a SNUR under TSCA.
SARA
CERCLA Hazardous Substances and corresponding RQs
None of the chemicals in this material have an RQ.
SARA Section 302 Extremely Hazardous Substances
None of the chemicals in this product have a TPQ.
Section 313
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No chemicals are reportable under Section 313.
Clean Air Act: This material does not contain any hazardous air
pollutants. This material does not contain any Class 1 Ozone
depletors. This material does not contain any Class 2 Ozone
depletors.
Clean Water Act: None of the chemicals in this product are listed as
Hazardous Substances under the CWA. None of the chemicals in this
product are listed as Priority Pollutants
under the CWA. None of the chemicals in this product are listed as
Toxic
Pollutants under the CWA.
OSHA: None of the chemicals in this product are considered highly
hazardous by OSHA.
STATE
CAS# 10102-17-7 is not present on state lists from CA, PA, MN, MA,
FL, or NJ.
California No Significant Risk Level: None of the chemicals in this
product are
listed.
European/International Regulations
European Labeling in Accordance with EC Directives
Hazard Symbols:
Not available.
Risk Phrases:
Safety Phrases:
S 37 Wear suitable gloves.
S 45 In case of accident or if you feel unwell, seek
medical advice immediately (show the label where
possible).
S 28A After contact with skin, wash immediately with
plenty of water.
WGK (Water Danger/Protection)
CAS# 10102-17-7: 0
Canada - DSL/NDSL
None of the chemicals in this product are listed on the DSL or NDSL
list. Canada
- WHMIS
This product has a WHMIS classification of D2B.
Canadian Ingredient Disclosure List
Exposure Limits
Section 16 - Additional Information
MSDS Creation Date: 12/12/1997 Revision #5 Date: 3/18/2003 The
information above is believed to be accurate and represents the best
information currently available to us. However, we make no warranty
of merchantability or any other warranty, express or implied, with
respect to such information, and we assume no liability resulting
from its use. Users should make their own investigations to determine
the suitability of the information for their particular purposes. In
no event shall Fisher be liable for any claims, losses, or damages of
any third party or for lost profits or any special, indirect,
incidental, consequential or exemplary damages, howsoever
arising, even if Fisher has been advised of the possibility of such
damages.
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