Download the use of chemical and microbial tracers in environmental studies

THE USE OF CHEMICAL AND MICROBIAL
TRACERS IN ENVIRONMENTAL STUDIES
In hydrology a tracer is defined as matter or energy carried by
water which will give information concerning the direction
and/or velocity of the water as well as potential contaminants
which could be transported by the water.
The first reported ground water tracing experiment was almost
2000 years ago when Philip, the tetrarch of Trachonitis, threw
chaff into a crater lake and reported that the chaff appeared downgradient in one of the springs at the headwaters of the Jordan
River. 2000 years have seen many changes occur in the
environment and in scientific methods and procedures.
The microbial contamination of ground water is a serious problem
that has resulted in large outbreaks of waterborne disease.
There are many sources of human pathogenic micro-organisms
which can cause ground-water contamination including leaky
sewer lines, sanitary landfills, waste water, etc..
The movement of bacteria and viruses into ground water is
influenced by many environmental factors which are difficult to
define completely.
Thus, the ability to trace microbial movement in ground water is
essential in recognising the potential for transmission of diseasecausing micro-organisms.
it is desirable to use tracer techniques to
• monitor the movement of microorganisms with percolating
water through the soil systems and ground water when
assessing new sites for land application of waste water,
• septic tank drainfields,
• investigation of sources of waterborne disease outbreaks.
An ideal ground water tracer is
• nontoxic,
• inexpensive,
• moves with the water,
• is easy to detect in trace amounts,
• does not alter the natural direction of the flow of the water,
• is chemically stable for a desired length of time,
• is not present in large amounts in the water being studied,
• is neither filtered nor sorbed by the solid medium through
which the water moves.
Almost certainly, the ideal ground water tracer does not exist.
Most ground water-water tracers used for hydrological and
geological studies are chemicals,
• most often fluorescein dyes or
• halogen salts.
They are used to determine
• directions and
• velocities of surface flow.
The criteria for selecting a suitable tracer organism for groundwater contamination is the duration of survival as well as their
retention in soil-water systems.
Microbial tracers have the advantage of
• not being mutagenic
• not having potential toxic effects, and
• having a finite lifetime.
TYPES OF MICROBIAL TRACERS
Bacteria are the most commonly used microbial tracers because
of their ease of growth and detection.
Coliform bacteria and in particular Escherichia coli are
extensively used for monitoring ground-water quality.
Coliform bacteria are excreted in large numbers in the faecal
wastes of man and other warm-blooded animals and thus can be
used to monitor the movement of septic and sewage wastes in
ground water.
As the faecal coliform bacteria are not natural inhabitants of soil
and ground water, they can be differentiated easily from common
soil microflora by their ability to grow on selective media at
elevated temperatures.
faecal coliforms were traced at a land disposal site in New
Zealand over a distance of 900m. The bacterial moved at a rate
of approximately 150 m/day.
Based on this rate of movement and the measured survival time
for the bacteria, faecal coliforms were estimated to be traceable
for at lease 2.5km from their source.
The disadvantage of using faecal organisms as tracers is the
difficulty in differentiating between those which derived from
the suspected source and those from other sources.
Therefore, faecal bacteria with specific characteristics i.e.
a marker which allows the tracer organisms to be distinguished
from all background organisms in the system should be used.
e.g.
antibiotic-resistant strains of E. coli and Enterococcus faecalis
are distinguished from naturally-occurring organisms by their
ability to grow on media containing antibiotics which suppress
the growth of the naturally-occurring organisms.
Bacillus stearothermophilus and H2S-producing strains of E. coli.
Can be used under limited conditions.
As both E. coli (H2S+) and Bacillus stearothermophilus can be
present in raw and treated effluent, they are unsuitable as tracers
in sewage polluted waters.
Tracer experiments have also been conducted by injecting a
mixture of antibiotic resistant E.coli strains into a well site and
monitoring their movements in down stream wells.
The rate of movement during this experiment was found to be 350
meters per day.
Other species of bacteria indicated to have potential as tracer
organisms include, Bacillus chromobacterium, Enterococcus,
Serratia marcescens, Chromobacterium violaceum and Bacillus
globigii as tracers through ground.
Caution. These organisms may naturally be present in the soil.
Enterococcus zymogenes was demonstrated successfully as a
ground water tracer in terms of its long term survival and
capability to move on ground water.
Bacteria have the following advantages as ground water tracers.
They are:
• easy to grow in large numbers and to assay.
• through the use of various markers they are usually
distinguishable from other flora likely to be present in the
ground water and the soil.
Caution in their use.
they could be potential pathogens;
E. coli has enterotoxigenic strains which can cause disease in
man. (E. coli O157:H7)
• Some m’orgs are capable of growth in the environment, may
produce erroneous results;
• many are large enough to be filtered out on certain soils;
• can adsorb to a variety of surfaces removing them from
circulation;
• their movement probably does not reflect the movement of
viruses.
• even with markers, like antibiotic resistance, it is often
difficult to distinguish test organisms from those naturally
present because markers can be lost during interaction with
natural populations.
It also would seem to be imprudent to inject antibiotic resistant
micro organisms into ground water because of the possible
transfer of resistance to potential human pathogens especially
when the water might be consumed later.
The odds of this can be reduced greatly by the use of bacteria
which do not carry the genetic information for antibiotic
sensitivity on plasmids.
Thus prudence should be used in the selection of bacterial species
to be used as tracers, and should be used only when experimental
conditions dictate or where the movement of ground water is
traced back to a source of pollution where organisms are already
present.
baker's yeast (Saccharomyces cerevisiae) has been used to trace
movement of microorganisms in artificially recharged ground
water.
351bs of yeast cells were injected into wells,
yeast cells were found to penetrate more than 7 meters into a sand
and gravel aquifer in less than 48 hours.
In evaluating the use of yeasts it should be noted that the distance
over which the yeasts cells were followed in these experiments
was short and their survival is dependent on nutrients present
in the environment.
The spores of the club moss, Lycopodium clavatum had been
used primarily by speleologists as tracers to determine
subterranean drainage characteristics in limestone regions.
The spores are small cellulose bodies 30 µm in diameter.
The technique involves colouring, Lycopodium spores with
biological strains, injecting the dye spores into the flow system at
sinkholes and trapping the spores with plankton nets at potential
resurgencies.
Spores are then examined under a microscope.
advantages of using these spores in karst regions.
• Their size and density reportedly allowed them to move
readily with ground water.
• They are resistant to detrimental factors in the environment
and appear to be '1harmless" non pathogens.
• In addition, they can be stained with five different colours of
biological stains permitting their used as multiple tracers.
Animal viruses, such as, hepatitis and the Norwalk agent are
known to be transmitted by contaminated ground water.
Other animal viruses pathogenic to man have been isolated
from ground water.
Because all human enteric viruses are capable of causing
disease they cannot be used safely as tracers.
To overcome this problem, vaccine strains of poliovirus type 1
has been used as a tracer but the vaccine strain of poliovirus
can be responsible for serious disease in man, although the
odds are extremely low.
Thus, caution must be observed carefully even if the vaccine
strain is used.
Most animal enteric viruses are not known to infect man. Thus,
bovine enterovirus type 1 has been used to trace the movement
of viruses from septic tank.
Bacteriophages i.e. a λ-like bacteriophage of E. coli K 12 was
used and found to be an excellent tracer of water movement,
especially in polluted rivers.
The advantages of using bacteriophages as water tracers are:-
• The phage is non-pathogenic to man and domestic animals;
• It is specific for its host bacteria;
• Assays, simple and rapid;
• It has good survival characteristics.
type 2 phage of Aerobacter aerogenenes 243 has been used to
follow the movement of shallow ground water in South Wales
over a distance of up to 680 meter.
Coliphage f2 has been used as a tracer virus to examine its
accumulation and movement in the soil and ground water
beneath the rapid infiltration land application site.
Both pathogenic animal viruses indigenous to sewage and tracer
f2 were detected in the ground water at a horizontal distance of
183 meter from the application site, indicating the potential use
of f2 phage for tracing movement of pathogenic animal viruses.
bacteriophages fit the criteria as ideal indicators most closely.
In addition, because of host specificity, phages can be mixed,
injected together, then distinguished on different hosts thereby
permitting simultaneous multiple tracers.
One drawback is that bacteriophages might be removed by
adsorption to the soil.
Methodology is available for the concentration of phages from
large volumes of water. With the use of these techniques it is
possible to detect as little as one to two phages in a 20L volume
of water.
Phage assay requires 12 to 24 hours which may be a drawback in
some applications, but for most studies would pose no difficulties
when samples are collected over long periods of time;
furthermore, samples can be frozen for long term storage and
assayed later.
Phages are also available which have no known host
common in the subsurfaces environment which can
support their replication. For the most part, however,
human and animal viruses are not suitable for tracer work
because of potential public problems.
CHEMICAL PHYSICAL TRACERS
Because water has a high specific heat capacity compared with
most natural materials, water does not change temperature
rapidly as it migrates in the sub-surface.
For e.g.
Temperature anomalies associated with the
spreading of warm waste water in the Hanford
Reservation in the U.S, for example, were detected
more than 5 miles from areas where the water entered
the sub-surface.
Changes of water temperature alter density and viscosity of the
water, which will alter the velocity and direction of flow of the
water.
e.g. injected ground water at 40°C will travel more than twice as
fast in the same aquifer under the same hydraulic gradient as will
water at 5°C.
Chemicals
i.e. common salt will almost entirely ionise when dissolved in
water.
ionisation increases the electrical conductivity of the water
containing the tracer.
Method has been used for 100 years.
However, high concentration of salt solutions must be introduced
as tracers in order to produce significant changes in the electrical
conductivity of the water at sampling points some distance away.
As concentrated solutions will be denser than the native ground
water they will tend to sink to the bottom of the aquifer and not
follow the natural flow paths of the water.
As a consequence, tracers are usually diluted so much in order to
reduce their density effects that specific analytical detection of
selected ions must be utilised rather than electrical conductivity.
The number of ion types which might be used is very large.
because of the low cost, ease of detection and low sorption,
chloride and bromide are most popular.
Because of sorption by cation exchange and the relatively high
natural abundances of most ions,
Br- is one of the best possibilities as a general tracer for ground
water studies.
nitrate ion can be used to monitor the percolation of the ionic
constituents of septic tank effluent in a range of soil types and
hydrogeological conditions.
Iodide has been successfully used as a tracer for surface water
and less so as a subsurface tracer.
Iodide tends to be sorbed to a greater extent than either bromide
or chloride.
Natural concentrations of iodide, however, are very low,
generally less than 0.01 mg/I, and methods for sensitive analysis
of iodide are relatively simple.
Chelating agents with heavy metals can be used to produce
complexes which commonly have a zero charge.
These complexes have low sorption on natural materials.
For e.g. EDTA.
heavy metals which have been used as tracers are
• copper,
• zinc,
• cobalt and
• lead.
Higher costs of chemicals and detection methods, together with
problems of toxicity have discouraged the widespread use of
heavy metals as tracers.
However, neutron activation analysis, while relatively expensive
can provide a very sensitive method for detection of many metals
thus allowing the use of tracers at extremely low dilutions.
Groundwater tracing using organic anions, i.e benzoate,
Sensitivity of detection and precision of measurement are very
good using liquid chromatography, and, like other anions, they
are highly mobile in natural aquifer systems.
Several naturally occurring stable isotopes of the common
elements have been used as ground water tracers.
i.e H,C,N,O,S
Requires
• special analytical equipment,
• tedious analysis,
• high total costs
have discouraged the use of most stable isotopes for artificially
injected tracers.
Stable isotopes, however, have been very useful for many
special hydrologic studies.
"Heavy
water"
(deuterium)
containing
relatively
large
concentrations of 2H has been used successfully as an injected
tracer.
increased awareness of radiation hazards has mitigated against the
use of radio active tracers,
may be used under certain conditions for the tracing of
nonpotable water which is isolated from surface.
One of the most commonly used radioactive tracer is 3H because
it forms part of the water molecule and travels with the ground
water. If it were not hazardous and if it were easier to detect
very small quantities it would be an ideal tracer.
Various organic dyes of low to negligible toxicity have
long been used as ground water tracers.
Because most of them are easily sorbed on solid materials,
these tracers are best adapted to tests involving short travel
distance, or within very permeable aquifers such as recent
basalt, gravel and cavernous carbonate rocks.
Advantages:
• low general toxicity,
• easy detection,
• high sensitivity and
• relatively low cost.
The most useful of the dyes can be detected
• visually in concentrations of a few parts per million,
• by fluorometers in parts per billion, and
• by sorption in activated charcoal and subsequent
desorption < 0.1 part per billion.
trace amounts of natural organic compounds may interfere
with the detection of such low concentrations.
Most tracer tests with dyes can be affected by
• decomposition by strong light,
• changes of pH and temperature
• and sorption on solid surfaces.
Tracer Dyes for e.g
fluorescein,
rhodamine WT (orange)
lissamine FF (green) and
amino G acid (blue) are among the most useful for water tracing.
Blue fluorescent dyes, which are used in the textiles and
paper industry to enhance the white appearance of these
products are called optical brighteners.
These dyes can be used as artificially injected tracers.
e.g. lucophor PVS.
Numerous gases have been used as sub-surface tracers but the
tracing has most commonly been of the movement of air in the
non-saturated zone.
Chemically inert, but radioactive
133
Xe and
85
Kr appear to be
quite suitable.
Ethylmercaptan, a gas with a strong odour has been used in karst
areas to establish the degree of inter-connection among various
cavernous openings.
One of the least expensive and most effective sources of
olefactory tracers gases which has been reported are
expired skunks.
Because of their non reactive and non toxic nature noble
gasses are attractive tracers.
Radon which is present in the subsurface, but owing to its short
half life (3.82 days) and the absence of parent uranium in the
atmosphere, is virtually absent in surface water which has reached
equilibrium with the atmosphere.
Surveys of radon in surface streams and lakes have, therefore,
been useful in detecting places where diffuse groundwater enters
surface waters.
Fluorocarbons compounds as chemical tracers.
They are extremely non reactive and
• do not break down chemically under normal ground water
conditions.
• They are easily detectable in water and in very low
concentrations with an optimum working range of between 1
and 100 ppt.
The limitation of fluorocarbons, however, is that
• they are organic compounds and as such they
• become dissolved in or
• strongly sorbed by many other organic materials,
therefore, they are unsuitable for tracing water in subsurface coal,
peat, oil, shale and other organic rich rocks.
comparative studies with the use of chemical and microbial
tracers.
i.e. antibiotic-resistant E. coli and fluorescein dye as tracers of
ground water flow in a soil series in Oregon.
The bacteria were recovered at the 45cm depth, 15m downslope
one hour after addition.
large numbers of E. coli survived during the 12 hour sampling
period.
Consideration should be given to more frequent use of marked
strains of bacteria as tracers since these organisms more closely
represent subsurface movement of microorganisms in soil and
groundwater.
A combination of chemical and biological tracer
materials
must
be
used
to
gain
an
accurate
representation of the movement specific pollutant types
in the effluent.
e.g. Bacillus globigii spores and a bromide ion tracer
would give an accurate indication of the risk of ground
water pollution from septic tank effluents in a range of
soil/overburden types and hydrogeological situations.
Bacillus globigii now known as B. atrophaeus.