Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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AUTOMATON FOR THE DETECTION OF POLLUTION IN AN
AQUEOUS MEDIUM IMPLEMENTING A TEST ON A I~PE OF MICROOR-
GANISM
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to the detection of pollution in an aqueous medium,
for example the detection of the pollution of water courses or discharged water and,
more particularly, to the field of automatic devices used for this type of detection.
2. Description of the Prior Art
Automatic stations for the detection and analysis of pollution, such as
stations for the protection of the inlets for drinking water production units comprise
devices that enable the automatic and continuous checking of a certain number ofphysical/chemical parameters such as the hydrocarbon, ammonia or heavy metal
content of the analyzed water. These devices enable the qualititative and
quantitative identification of these different types of polluting agent.
As a complement, these stations also include installations that can be used
to obtain a detection of pollution even when there is no identification of the
pollution agent, and that are provided with warning means that can be triggered
when massive pollution, taking the form of severe toxicity of the analyzed water, is
detected.
Devices such as these carry out tests on living organisrns. These tests
consist in observing the modifications in the metabolism of these organisms in the
presence of the analyzed water during a determined period of time. The biological
criteria taken into account to observe the modifications of the metabolism are
generally the phenomenon of death or any other modification that can be noted
visually. The value of such tests lies in the fact that their interpretation from the
sanitary viewpoint is immediate since they consist in observing the response of a
living organism to the quality of its environment. The result obtained is therefore
comprehensive and represents all the complex reactions (such as the effects of
synergy or antagonism) that take place within the aquatic ecosystem and that cannot
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be perceived through physical/chemical analysis.
In being based on a non-specific criterion, namely the toxicity at the level
of an organism, these tests offer the user the possibility of tracking down a greater
number of cases of pollution.
Several types of tests on living organisms have been devised to this effect.
The most frequently employed tests consist in observing the survival of Brachydanio
rerio, Daphnia magna (the "Daphnies test") or Colpidium campylum in a sample of
monitored water. The AFNOR standard T90-301 thus recommends the use of the
microcrustacean Daphnia magna (Daphnies test). These tests require an exposure
time of the order of 24 hours which is necessary for a reliable interpretation of the
response of the observed organism to its aquatic environment.
However, tests such as these have the drawback wherein they cannot be
carried out in automatic permanent checking installations.
Other tests, which can be carried out continuously, are therefore used.
Thus, in one test, trout are placed in a container in which there flows a current of
water to be monitored. The death of the trout, which reflects a state of severe
pollution of the water, is detected by means of an appropriate device. However, this
test is not frequently used owing to its low sensitivity which makes it inapplicable
to the control of water pollution beyond a certain level of toxicity.
Other tests, using microorganisms, have therefore been designed. These
tests have a sensitivity comparable to that of tests using Daphnies, and furthermore
have the advantage wherein they can be automated.
Tests of this type have many advantages, notably that of using microorga-
nisms which can be stored in Iyophilized forrn and therefore ha~e long preservation
times.
One of the most efficient tests that uses microorganisms consists in
observing the intensity of light output from luminescent bacteria present in thewater to be analyzed in relation to the intensity of light output produced by the
same quantity of luminescent bacteria present in a same quantity of reference
3Q water. The fall in intensity of light output, proportional to the fall in the metabolism
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of the bacteria, can be used to assess the toxicity of the analyzed water.
Thus, Phosphobacterium phosphoreum is used. This is a marine bacterium
which has the property of emitting photons naturally in the course of its metabo-
lism.
S The test of inhibition of bacterial luminescence has numerous advantages
as compared with other tests of toxicity, notably as regards speed, sensitivity and
reliability. Indeed, while the conventional tests such as the Daphnies test call for a
response time of about 24 hours, the test of inhibition of bacterial luminescence can
be used to obtain a result in less than one hour, and this period includes the
preparation and measurement phases. The sensitivity of this test is moreover close,
in terms of performance, to that of the Daphnies test, and it has far greater
reproducibility.
Furthermore, each test is carried out on about 20,000 bacteria. Its result
thus offers true statistical representativity.
This type of test furthermore has the advantage wherein it can be
irnplemented in automated installations that can operate without human interven-tion.
For, the microorganisms can be stored in Iyophilized form so that the
installation has an available store of microorganisms enabling independent
operation for a period that may go up to seven days. These installations can
therefore be provided with remote transmission means by which, in the event of adetection of pollution, the warning may be triggered by remote control.
The use of lyophilized strains furthermore has the advantage of eliminating
the large number of constraints related to the setting up of a culture or of a
permanent breeding program necessary, for example, in the case of the Daphnies
test.
However, the use of strains of microorganisms in Iyophilized form to carry
out these tests raises problems related to their reconstitution, namely the operation
in which the metabolism of these microorganisms is activated, this metabolism
having been suspended by the Iyophilization operation. When the test is done, the
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strain should indeed be reconstituted according to criteria that enable the test to
be given a high degree of reproducibility. This operation of reconstitution generally
takes place as follows:
- the flasks containing the Iyophilized bacterial strain, preserved at a
temperature of 4C to 8C, are opened;
- the reconstitution liquid which may be formed, for example, by distilled
water with the addition of sodium chloride, is introduced into the flask;
- the flask is closed again and shaken so as to prompt the mixing of the
reconstitution liquid with the Iyophilized bacterial strain;
- the bacteria solution thus reconstituted can then be stored and displays
an activity that enables it to be used within a period of time which varies according
to the bacteria used, this period of time being of the order of 12 hours for
Phosphobacterium phosphoreum.
It has been obsened that this last step of the mixing of the Iyophilized
bacteria is decisive for the efficient reproducibility of the test. The studies that have
been carried out on this subject have led to seeking the homogenization of the
mixture by shaking it for example with a VORTEX type vibrator.
However, the use of the VORTEX machine leads to a destruction of a part
of the bacteria strain contained in the shaken mixture. Since the vibrations of a
device such as this are not absolutely reproducible, this deficiency of reproducibility
causes the rate of destruction of the strain to vary according to the samples and
affects the very reproducibili~y of the test.
SUMMARY OF THE INVENTION
An aim of the invention is to propose an automaton for the detection and
quantification of the pollution in an aqueous medium, implementing a test on a type
of microorganism and having means that enable the Iyophilized strain of the
microorganism used to be mixed with a reconstitution liquid according to a method
that enables the test to be given high reproducibility.
Another aim of the invention is to provide an automaton with means such
as these, incapable of even partially destroying the bacterial strain used.
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These aims as well as others are achieved through the invention, the object
of which is an automaton for the detection of pollution in an aqueous medium,
implementing a test on a type of microorganism and comprising means enabling thereconstitution of the Iyophilized strain of said microorganism by mixing it in at least
one container with a reconstitution liquid. The containers may be constituted byflasks void of air, in which said Iyophilized strain is conditioned.
According to the invention, said automaton comprises means for the mixing
of said strain with said reconstitution liquid by the overturning of said container,
said means for mixing by overturning making it possible to avoid causing even the
partial destruction of the strain. In the present text, the term "overturning" is
understood to mean the movement of total or partial rotation of said container in
a plane that is vertical or substantially vertical along an axis that is perpendicular
or substantially perpendicular to its longitudinal axis.
According to a preferred variant, said means of mixing by overturning are
constituted by a shaft that is fixedly joined perpendicularly to a rack of said
container or containers, said shaft being connected to means enabling it to apply
a rotation on itsel
Advantageously, said rack is provided with thermo-regulation means that
enable the temperature of the containers placed therein to be kept constant.
Preferably, this temperature corresponds to the temperature of preservation
of the microorganism.
Also preferably, said means enabling the application of a rotation to said
shaft are constituted by a gear system including a link-rod.
Advantageously, said means for the mixing by overturning of said container
or containers enable the communicating, to said containers, of an overturning
motion of at least 45 in both directions of overturning.
Also advantageously, said shaft is provided with a support for the feeding
pipes conveying the thermo-regulation fluid into said containers.
According to a promising feature, said support has a spiral configuration
enabling said feeding pipes to be wound about said shaft without hampering the
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rotation of this shaft.
Although the invention can be implemented for every type of pollution
detection automaton using a test on a type of microorganism lyophilized before-
hand, it preferably concerns an automaton that carries out a test consisting in
detecting the drop in light intensity corresponding to a fall in the metabolism of the
luminescent bacteria under the influence of a toxic effect. In this type of test, the
measurement of light intensity is indeed carried out by means of a photomultiplier.
The mixture of the Iyophilized strain and the reconstitution liquid should therefore
be done in a manner that is extremely reproducible with the aim of not affectingthe operation carried out by the photomultiplier.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention as well as its different advantages shall be understood more
easily from the following non-restrictive embodiment, made with reference to theappended drawings, of which:
- Figure 1 shows a general drawing of the different constituent elements
of an analyzer enabling the severe toxicity of water to be tested by inhibition of the
bacterial luminescence;
- Figure 2 shows a graph expressing the principle of the test by inhibition
of the bacterial luminescence;
- Figure 3 shows a graph as obtained by means of the analyzer according
to figure 1;
- Figure 4 shows a top view of a part of the analyzer according to figure
l;
- Figure S shows a front view of the gear system permitting the mixture of
the Iyophilized strain with the reconstitution liquid.
MORE DETAILED DESCRIPTION
According to figure 1, an automaton for the detection of pollution in an
aqueous medium that can be used to check the toxicity of surface water, industrial
effluents, seepage water or, again, domestic waste water, comprises:
- a manipulator arm that can shift a needle along three axes X, Y, Z and
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is fitted out with a micro-syringe (1);
- a compartment refrigerated at 4C for the preservation of the flasks
containing the Iyophilized bacterial strain and the reconstitution of the bacterial
strain by the addition of the reconstitution liquid (2);
S - a refrigerated compartment for the preservation of solutions of
reconstituted bacteria (3);
- a thermo-regulated compartment at the working temperature to prepare
the samples to be tested and the reference water and to place them under the
requisite temperature (4);
- a measurement unit including a photomultiplier and enabling the
measurement of the light output of the bacteria in the presence of the substances
contained in the monitored water as compared with the light output of the bacteria
of a reference sample (S);
- a microcomputer (6) working by means of a program (7) managing the
different movements of the manipulator arm, enabling the computation of the
inhibition of bioluminescence, the storage of different data and the printing ofgraphs and results on a printer, and capable of carrying out the remote transmission
of a warning signal to a factory (9). The program (7) also includes instructions that
can be used, if necessary, to adjust and control the different steps of the testmanually.
According to figure 2, the following is the principle of the test used by the
detection automaton shown in figure 1.
A marine bacterium (Photobacterium phosphoreum) is capable of naturally
emitting a light under conditions favorable to its metabolism. This bacterium can
be preserved for a long period of time in Iyophilized form. In the presence of atoxic substance, the bacteria cells reduce their light output7 sometimes to the point
where this output is totally extinguished. During the test, two tubes are inoculated
with an identical quantity of bacteria. One of the tubes contains non-toxic reference
water while the other tube contains the water to be tested. The progress of the light
output (L) as a function of time (T) is then tracked for 30 min (curves A and B)
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at a working ternperature of 15C. The curve A expresses the progress in time ofthe light output of the bacteria contained in the reference sample and the curve B
expresses the progress, during the same period of time, of the light ou$put of the
bacteria contained in the sample of monitored water. If, at the end of 30 min, the
S difference between the intensity of light output of of the reference sample and the
intensity of light output of the tested sample is greater than a certain threshold, the
tested sample is considered to be toxic. The response time is directly related to the
toxicity of the product and to its concentration. The diminishing of the biolumines-
cence in the tested sample may therefore, depending on each case, be observed
from the very first seconds when the bacteria solution is put into contact with the
polluted water, or it may be observed far later. This test can thus be used to obtain
a quantitative response expressing the degree of toxicity.
The simplicity and speed of the method give this test undeniable
advantages which explain the fact that its field of application has swiftly beenextended to cover the checking of water (inland water and sea water) pollution as
well as to the study of the severe toxicity of chemical products.
According to figure 3, the analyzer according to figure 1 has been used to
test the toxicity of river water. The curve C expresses the progress in time of the
decrease in the bioluminescence of bacteria placed in the presence of this water,
namely the difference in light output observed after 30 min between the tested
sample, containing the monitored water, and the reference sample. The test was
carried out every hour for 24 hours. The curve C of results reveals a crossing of the
danger level or warning threshold between 13.00 and 19.00 on the one hand and
between 21.30 and midnight on the other. The warning may be displayed on the
screen of the microcomputer 3 and then remote transmitted to a drinking water
production unit located downstream from the station in which the automaton is
installed.
In figure 4, the analyzer comprises a frame 10 on which there is mounted
a manipulator arm 11 provided with a needle 12. The arm 11 can be used to shift
the needle 12 along three axes. The manipulator arm is connected to a micro-
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syringe, not shown in the drawing, that enables the manipulation of the different
liquids necessary for the test. A rack 13, thermo-regulated at 4C, is mounted inside
this frame 10 and has receptacles in which there may be placed flasks 14 containing
a determined quantity of a Iyophilized strain of Phosphobacterium phosphoreum.
The rack 13 is furthermore fitted out with overturning means 15 constituted by ashaft 16 which is fixedly joined to it perpendicularly, it being possible for said shaft
16 to be put into rotation by means of a gear system 17 controlled by a motor 18b.
The shaft 16 is fitted out with a support 18 that has a spiral configuration. This
support 18 can be used to receive feeding pipes 19 for the cooling liquid of theregulation system of the rack 13 and makes it possible to avoid hampering the
circulation of th;s liquid during the rotation of the shaft 16.
The analyzer also comprises a rack 20 for the intermediate storage of the
reconstituted bacteria suspension fitted out with thermo-regulation means by which
its temperature can be maintained at 4C.
A third rack 21 can be used to receive tubes in which the water to be
tested and the samples are brought to the required temperature. To this effect, the
water to be monitored is fed in continuously to be made available in a well 22.
A measurement unit 23 comprises a photomultiplier facing which there is
positioned a barrel provided with two tubes designed to receive the bacterial
suspensions, one lot of bacterial suspension with the sample and the other with the
reference water. This device can be used to follow the progress of the biolumines-
cence of each of the two solutions without any manipulation other than the rotation
of the small barrel.
According to figure 5, the gear system 17 by which the shaft 16 can be
made to rotate comprises a toothed wheel 24 fixedly joined to the shaft 16
combined with a second multiplier toothed wheel 2S. A link-rod 26, actuated by the
motor 18, is used to put the wheel 25 into rotation and thus to induce the putting
into rotation of the shaft 16 by transmission through the toothed wheel 24.
The device works as follows.
The rack 13 is loaded with fourteen flasks 14, each containing a Iyophilized
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sample of Phosphobacterium phosphoreum. These flasks 14 are closed by a cap thatcan be pierced by the needle 12 actuated by the manipulator arm 11. The racks 20and 2 are each loaded with 16 empty tubes. During the use of the device, the arm11 gets shifted in such a way as to position the needle 12 on one of the flasks 14
S and perforate the cap of said flask so as to inject therein 2 ml of the reconstitution
liquid formed by a solution of 2% sodium chloride in distilled water. The
salification of the reconstitution liquid is indeed necessary to balance the osmotic
process. The handling arm 11 then withdraws the needle 12 from the flask. The
motor 18 then allows the shaft 16 to be put into rotation by means of the gear
system 17 and the link-rod 26. The put~ing of the shaft 16 into rotation prompts the
complete overturning of the rack 13 in the first overturning direction and then in
the second overtuming direction.
This operation can be used to bring about the m*ing of the Iyophilized
strain with the reconstitution liquid in mild conditions that are no~ likely to cause
even the partial destruction of the strain. The bacteria population in the reconstitu-
tion liquid is then equal to 108 per ml.
The manipulator arm 11 then gets positioned at the flask 14 in which the
mixture was made so as to enable the needle to draw off the contents of this flask
14. These contents are then conveyed towards one of the tubes of the rack 20. Since
the rack 20 is thermo-regulated at 4C, this tube enables the sts)rage of a quantity
of reagent that can be used for 12 hours.
The manipulator arm 11 then prepares the water, for testing, in two tubes
of the rack 21. 900 ~l of the sample taken from the well 22 are raised to a content
of 2% with 100 ,ul of NaCI at 20%. 900 ~l of distilled water is taken to 2% by
means of 100 ,ul of NaCI at 20% to constitute the reference. The two samples of
water are taken to the temperature of 15C at which the test is carried out. A
solution of NaCI is also prepared in one of the tubes of the measuring unit 23. The
manipulator arm then transfers 10 ,ul of the bacteria reagent stored in the rack 20
as well as 90 ~1 of NaCI at 2% into each of the tubes of the measuring unit 23.
After the measurement of the initial lurninescence, the 900 ~l of the sample and of
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the reference water coming from two tubes of the rack 21 are transferred to eachof the tubes of the measuring unit. The operation continues with the simultaneous
acquis;tion of the bioluminescence of each of these tubes at every minute for 30min.
S The decrease of the luminescence of the sample containing the water to
be analyzed in relation to the luminescence of the reference sample is computed
each minute. The graphs of luminescence and of decrease obtained are displayed
on the monitor of the microcomputer 3 and, at the same time, are plotted on the
printer 7. These graphs may be interpreted by a technician who may raise the alarm
if the decrease measured goes beyond a certain threshold. At the end of the test,
the manipulator arm 11 carries out the draining and rinsing of the used tubes
before a new test is started.
Since the number of flasks, containing the Iyophil of Phosphobacterium
phosphoreum, placed in the rack 13 enables the device to be fully autonomous forseven days, this device can work without any human presence throughout this
period. Cases when the threshold of decrease is crossed are then expressed by the
triggering of a remote-transmitted alarm.
In a warning period, the frequency of the test may be accelerated so as to
follow the progress of this decrease with greater precision.
The aspects of the present exemplary embodiment of the invention as
specified here above are in no way exhaustive. In particular, it is possible, without
departing from the spirit of the present invention, to envisage the use of othermeans of overturning the container or containers to carry out the reconstitution of
the strain of the microogranisms without causing ~he even partial destruction of this
strain.
