FR3078075A1 - METHOD OF ESTIMATING GLOBAL TOXICITY OF AQUEOUS SAMPLE, KIT FOR ITS IMPLEMENTATION AND USE FOR VARIOUS TYPES OF EFFLUENTS - Google Patents

Abstract

Method for estimating the overall toxicity of an aqueous sample comprising the following steps: - distribution of different fractions of the aqueous sample of a test medium in different measuring cells each containing a single microbial strain, non-pathogenic, belonging to a panel of microbial strains exhibiting decreasing toxicological resistance profiles, each strain being previously assigned a toxicological resistance index with respect to molecules or conditions frequently encountered in said test medium; monitoring, in a thermostatically controlled chamber, the inhibition of the microbial respiratory activity of each sample fraction for a given duration, the final result and / or the kinetic results relating to each fraction, providing information on the activity respiratory of microorganisms, - determination of an overall toxicity index of the aqueous sample taking into account the indices of robustness of each of the strains. Kit for implementing said method. Use for natural waters or wastewater treatment plants or industrial effluents.

Description

FIELD OF THE INVENTION

The present invention relates to a method for evaluating the toxic nature of an aqueous effluent, a kit for the implementation of said method and its use in the field of the purification of domestic, agricultural or industrial water and environmental monitoring. .

PRIOR ART

The evaluation of the toxicity of an aqueous sample (industrial effluent or urban wastewater for example) consists in bringing this sample into contact with a given organism under determined experimental conditions in order to observe the effects on the activity of said organism. However, this parameter is directly dependent on the organization used.

Numerous evaluation tests have emerged commercially, each based on its own organism. V. Kokkali et al. published in 2014 (Trends in Analytical Chemistry 61, 133-155) a review of commercial tests dedicated to measuring the toxicity of aqueous samples. In 95% of cases, the tests are based on mono-parametric approaches using a single organism. The main limitation of these tests lies in the representativeness of the information collected. Indeed, each organism provides information on the effect of a sample on an aspect of its own metabolism (respiratory activity, cell mortality, growth rate, ...). Consequently, the representativeness of the information collected is limited.

In order to overcome this limitation, the company NCIMB (UK) offers two similar methods (MARA and LumiMARA) for the evaluation of the toxicity of a chemical compound, based on a panel of controlled microorganisms. In the case of the MARA test (Microbial Assay for Risk Assessment), ten bacterial strains and one yeast strain constitute the biological panel. This test aims to measure the toxic effect of said compound at different concentration gradients on cell growth and to compare the response obtained with profiles previously established for known chemical compounds (see also patent EP1283899B1). The limit of the interpretation of this MARA test lies in the analysis of the multivariate data collected, which does not take into account the robustness of the strains used. A very sensitive strain with the same weight in data analysis as a more robust strain. Therefore, a sample inducing toxicity on the most robust strain will be considered to have a toxicity similar to a sample inducing an effect on the very sensitive strain.

In the case of the LumiMARA test, these are only bioluminescent bacterial strains, the test focusing on the inhibition of bioluminescence in order to estimate the toxic effect. This test has the same drawbacks as the MARA test. In addition, most of these bioluminescent bacterial strains are of marine origin, which requires specific preparation of the samples to be tested with lower salinity than seawater (in particular adjustment of salinity).

Most of the current measurement methods lack representativeness vis-à-vis the overall toxicity parameter of an aqueous sample.

GOALS OF THE INVENTION

A first aim of the invention is therefore to propose a method making it possible to assess the overall toxicity of an aqueous sample and in particular making it possible to be able to compare samples with each other.

Another object of the invention is to propose a method for evaluating the toxicity of an aqueous sample which is precise, reproducible and rapid (preferably less than two hours), making it possible for example to pass on the information on conditions treatment or purification of the effluent tested.

Another object of the invention is to propose a method making it possible to assess the overall toxicity, without requiring dilution of the sample.

Another object of the invention is to propose a method which is easy to implement and which can be automated.

DESCRIPTION OF THE INVENTION

To this end, the present invention relates to a method for estimating the toxicity of an aqueous sample comprising the following successive steps:

distribution of different fractions of the aqueous sample of a medium to be tested in at least eight measurement cells, preferably at least ten measurement cells, each containing a single, non-pathogenic microbial strain, said microbial strains belonging to a panel different microbial strains with decreasing toxicological resistance profiles, each microbial strain being previously assigned a coefficient called robustness index or toxicological resistance index (Ind-Rob) vis-à-vis molecules or conditions frequently encountered in said medium to be tested;

- placement of measurement cells in a thermostatically controlled enclosure;

- monitoring of the inhibition of the microbial respiratory activity of each fraction of sample for a given duration, of at least 15 minutes, preferably at least one hour, the final result and / or the kinetic results relating to each fraction , providing information on the respiratory activity of microorganisms,

- determination of an overall toxicity index (Ind-TOX) of the aqueous sample in the presence of each microbial strain, taking into account the robustness indices of each strain.

It turns out that this method makes it possible to obtain a very rapid estimate, in less than two hours, or even as early as 15 minutes, of the overall toxicity of a sample, which allows for example a monitoring (almost in real time) of the treatment quality of a treatment applied to this effluent.

The estimation of the response of each microbial strain is taken individually at first, each individual response is then weighted by the robustness index of the microbial strain considered to obtain a value for the overall toxicity index.

Monitoring means, for example, a measurement at regular intervals of the toxic effect of the sample analyzed on the biological activity of microorganisms. The final result gives information on the ability to inhibit microbial growth of the sample and the slope of the curve, information on the speed of this inhibition with the microbial strain present.

According to a first embodiment of the invention, the monitoring of the metabolic activity of the microorganisms is carried out by means of an oxygen, redox or pH sensor present in the measurement cell.

According to a second embodiment of the invention, a marker for the metabolic activity of bacteria, such as a fluorescent and / or colored marker for the respiratory activity of said microorganisms is added to each measurement cell, the monitoring of the respiratory activity of said microorganisms being carried out by spectrophotometry.

The marker for the metabolic activity of bacteria is advantageously resazurin. Indeed, the resazurin molecule is reduced to pink-colored resorufin in the presence of cellular metabolic activity, this reaction being quantitatively correlated to the microbial respiration of the sample considered. With this marker, the monitoring of metabolic activity can be carried out by colorimetry (resazurin absorbance peak: 601 nm / resorufin absorbance peak: 571 nm) and / or by fluorescence spectrophotometry performed at a length excitation wavelength of 540 nanometers and an emission wavelength of approximately 610 nanometers.

The method according to the invention thus makes it possible to observe the kinetics of microbial growth, without requiring dilution of the sample.

This method can easily be automated.

Control measuring cells can be provided:

- Sample control (sample only + marker). This control makes it possible to measure the behavior of the sample on the marker without the action of microorganisms. This control must be deduced from the biological signal measured (microorganisms + sample + marker: TEST):

TEST - Sample control = real TEST

- Microorganism control (microorganisms + marker) is used as a biological reference measuring the activity of each of the strains of microorganisms under normal conditions (no toxicity). This control serves as a reference for calculating the inhibition rate (Tx Inh):

Tx Inh = (Microorganism control - real TEST) / Microorganism control

Preferably, each measurement cell is prepared according to the following successive steps:

- isolation and culture of the selected microbial strain, and implementation thereof in said cell,

- conditioning in said cell with addition of a protective agent for the microbial strain against lyophilization,

- then freeze-drying of the microbial strain and conservation.

The stages of culture of the microbial strain make it possible in particular to place the microorganisms in exponential growth phase with a homogeneous population.

The protective agent is advantageously a diholoside such as sucrose or a triholoside such as raffinose.

The lyophilization step is preferably preceded by a freezing phase in a temperature range from -20 ° C to -80 ° C, the lyophilization taking place at a temperature below -40 ° C under a pressure below 0.10 mbar (for example around 0.05 mbar).

For example, the different microbial strains selected, in particular for a toxicity assessment of a sample of effluent from urban or industrial wastewater or natural water, are part of a panel of bacterial strains chosen from: Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Enterobacter helveticus, Proteus mirabilis, Budvicia aquatica, Zoogloea resiniphila, Rhizobium radiobacter, Thauera linaloolentis, Bosea vertrisii, Comamonas testosteroni, Comamonas denitrificans, Arthrobacter aurescens, Staphylococcus sp, Acinetobacter towneri, Rhodococcus rhodochrous, Corynebacterium pseudodiphtheriticum , Variovorax paradoxus, Staphylococcus auricularis, Sphingobium yanoikuyae, Cupriavidus necator, Paracoccus denitrificans, Bacillus subtilis subtilis, Paracoccus pantotrophus, Bacillus funiculus, Bacillus mycoides, sorted in decreasing order of resistance to toxicity.

The individual results of the metabolic activity measurements such as the kinetics of respiratory activity of each of the strains, relative to a control, are expressed in inhibition rate (Tx_lnh) of said strain, and said individual results are grouped together giving a toxicological imprint of the sample.

Then, each microbial strain being initially assigned a coefficient called robustness index or toxicological resistance index (ranging from 0 to 1), an overall toxicological index of the sample is calculated according to the following formula (I):

_{,, rrnv} Zn = i (Jnd_Rob _n xTx_lnh _n ) lnu_T0X = ---————————--% n = l ( ^Ind - ^Rob n) (I) with x: number of microbial strains tested lnd_TOX: Overall toxicity index lnd_Rob _n : Robustness index of the strain n

Tx_lnh _n : Inhibition rate of strain n

The robustness index or toxicological resistance index is determined beforehand for each microbial strain, with respect to particular conditions (for example pH, salinity) or in the presence of toxic compounds (such as oxidants, disinfectants, antibiotics, heavy metals ...) likely to be encountered in surface water, effluent from treatment plants or in a particular industrial effluent to be tested.

This index makes it possible to weight the response obtained with each of the microbial strains. Indeed, among the microorganisms selected, some have a particularly strong tolerance towards the effluents to be tested while others, on the contrary, are immediately impacted by the compounds present in the effluents tested. The overall toxicity index thus takes into account the behaviors and sensitivities of each of the strains.

Thus by means of the method according to the present invention, the microorganisms selected not only provide a specific imprint of the toxicity of the test sample, but also an overall index of toxicity after weighting the responses as a function of the specific sensitivity of the microorganisms.

The present invention also relates to a kit for carrying out the evaluation of the toxicity of an aqueous sample, said kit comprising:

- at least eight measurement cells, preferably ten measurement cells, each containing a single microbial strain, non-pathogenic, and lyophilized in the presence of a protective agent,

preferably a control cell for the sample and a control cell for each microbial strain,

- a possible marker of metabolic activity of said bacteria, such as a fluorescent and / or colored marker, and

- detailed instructions for implementing the method as described above.

The measuring cells are advantageously closed glass vials, preferably in the form of tubes, individual tubes or arranged in microplates.

The present invention also relates to the use of the estimation method described above, for the estimation of the toxicity of an effluent of urban wastewater, effluent from the food industry or from industry. , stormwater, surface water, surface or groundwater, or marine water.

The method according to the invention finds a particularly advantageous use for monitoring treatment or purification channels, or environmental monitoring.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be clearly understood on reading the following description of exemplary embodiments, with reference to the appended drawings in which:

Figure 1 is a diagram showing the toxicological resistance of different bacterial strains.

Figure 2 shows examples of kinetics of respiratory activity of microorganisms as a function of time depending on the intensity of the toxicity.

FIG. 3 shows an example of monitoring the treatment of different effluent samples (A to E) with their inhibition rates of bacterial respiration with respect to different strains, before treatment and after treatment in a treatment plant (effluent F).

Figure 4 shows the overall toxicity indices calculated for the samples in Figure 3.

EXAMPLES

The different stages of preparation of the microbial strains before their introduction into the measurement cells are presented below:

Step 1: Culture of the strains before lyophilization

The selected microbial strain is isolated on a petri dish (LB agar medium: 5g / L NaCl, 5g / L Tryptone, 0.5g / L Yeast extract, 12g / L agar) at 30 ° C for 24 to 48 hours . It is then inoculated and incubated in LB medium (5g / L NaCl, 5g / L Tryptone and 0.5g / L Yeast extract) at 30 ° C, with shaking at 250 rpm for 16 to 20 hours. The culture is monitored by optical density (cell density indicator) at 620 nm, until a homogeneous culture of bacteria in the exponential growth phase is obtained.

Step 2: recovery and washing of the strains

After centrifugation (6400 revolutions per min / 4 min / 4 ° C), the pellet is recovered and optionally washed several times with a 10 ' ² M MgSCb solution, then conditioned in a lyophilization solution containing a protective agent and a carbon substrate . The protective agent here is raffinose. The carbon substrate can be:

- a specific carbon source (glucose, sodium acetate, pyruvate, ...)

- a known mixture allowing cells to have significant biological activity (wastewater standardized according to OECD standard 209).

Step 3: Lyophilization of the strains

The lyophilization solution containing said bacteria in suspension are distributed equally in different tubes, which are then subjected to freezing at -80 ° C for at least 3 hours, then to a lyophilization step at -50 ° C under 0, 05 mbar for 36 hours. The whole can then be stored for several months at -20 ° C.

Step 4: Preparation of the analysis

Freeze-dried bacteria can be rehydrated for 0-30 minutes by adding distilled water.

Each tube containing a unique microbial strain is then ready for the actual analysis.

Step 5: Analysis

The aqueous sample to be tested is added either directly to the lyophilized microbial suspension or to the rehydrated microbial suspension. Then, the metabolic activity indicator, here resazurin, is introduced into the measurement cell in the following proportions: 10% of marker (resazurin),

23.3% of microbial suspension and 66.7% of sample to be tested.

The monitoring of microbial respiration is then monitored by fluorescence (excitation wavelength of 540 nm and emission wavelength of 610 nm). The results for each strain take account of the controls carried out simultaneously (one control per bacterial strain tested = bacterial strain + marker and one control = sample + marker) as indicated previously in the description of the invention.

Each microbial strain selected gives partial information. However, to obtain a more global estimate of the biodegradable organic matter content of a sample, it is necessary to combine information from several microbial strains.

Selection of microbial strains

To this end, the inventors selected 28 bacterial strains (see Table 1 and Figure 1) from the natural environment (and frequently encountered in urban wastewater) and compared them to different molecules (such as antibiotics, heavy metals, disinfectants. ..) or different conditions (pH, salinity ...) that can affect their growth (that is to say potentially toxic for these bacteria). At least 21 different tests have been performed for each of the bacterial strains.

strains Collection Acinetobacter lowneri DSM 14962 Arthrobacter aurescens DSM 20116 Bacillus funiculus DSM 15141 Bacillus mycoides DSM 299 Bacillus subtilis subsp. subtilis DSM 10 Bosea vestrisii DSM 21229 Budvicia aquatica DSM 5075 Comamonas denitrificans DSM 17887 Comamonas testosteroni DSM 38 Corynebacterium pseudodiphtheriticum CIP A105 Cupriavidus necator DSM 428 Enterobacter helveticus DSM 18396 Escherichia coli ATCC 700926 Paracoccus denitrificans DSM 1404

DSM: German Collection of Microorganisms

ATCC: American Type Culture Collection

CIP: Collection of the Institut Pasteur

strains Collection Paracoccus pantotrophus DSM 65 Proteus mirabilis CIP A235 Pseudomonas aeruginosa DSM 11634 Pseudomonas aeruginosa DSM 6279 Pseudomonas putida DSM 1868 Pseudomonas putida DSM 84 Rhizobium radiobacter DSM 7215 Rhodococcus rhodochrous DSM 6427 Sphingobium yanoikuyae DSM 6900 Staphylococcus auricularis DSM 20609 Staphylococcus sp. DSM 1057 Thauera linaloolentis DSM 12138 Variovorax paradoxus DSM 645 Zoogloea resiniphila DSM 14766

Table 1

From all the results obtained, an index of toxicological resistance also called the robustness index (lnd_Rob) was assigned to each of the bacterial strains tested and these strains were classified in decreasing order of resistance to toxicity (see Figure 1 ).

For the toxicity tests of aqueous samples a panel of eight or ten bacterial strains with varied resistance indices (ranging between the values 1.00 for E. coli and 0.13 for B. mycoids) can thus be chosen .

For exemple

The panel used consisted of the following eight bacterial strains (with their toxicological resistance index):

Escherichia coli (1.00)

Pseudomonas putida (0.88)

Pseudomonas aeruginosa (0.81)

Rhizobium radiobacter (0.63)

Comamonas denitrificans (0.53)

Staphylococcus auricularis (0.38)

Bacillus subtilis subtilis (0.34)

Bacillus mycoides (0.13).

The inhibition rates of different samples of wastewater to be treated (noted A, B, C, D and E in Figures 3 and 4) entering a treatment plant were measured after monitoring the respiratory kinetics of each. of the eight strains individually for 1 hour 15 minutes at 30 ° C.

The effluent (F) leaving the treatment plant, after (physicochemical) treatment, was also tested in the presence of each of the eight bacterial strains individually.

The results grouped in FIG. 3 show for each sample an imprint of specific toxicity specific to the sample tested. It is noted that the fingerprints differ significantly between the samples demonstrating a toxicity induced by different chemical compounds.

The higher the inhibition rate, the greater the toxic effect on a bacterial strain.

The treatment applied significantly "reduces" the toxicity of the effluent vis-à-vis almost all of the strains.

Beyond the toxicological imprint which provides information regarding each of the strains, the overall toxicity index was determined by means of the following formula (I):

Ind_T0X =

J ^ _{= i} (J.nd_Rob _n X TxJ.nhj /)

Zn = lC ^Ind - ^Rob n) which, applied to the particular example of sample A:

(1 x 0% + 0.88 x 0% + 0.81 x 0% + 0.63 x 6% 4- 0.53 x 0% + 0.38 x 0% + 0.34 x 0% + 0 , 13 x 99%) / nd TOX = -_____________-_______________-_______________-_______________-_______________-_______________-_______________-____________ (1 + 0.88 + 0.81 + 0.63 + 0.53 + 0.38 + 0 , 34 + 0.13) gives an overall toxicity index IndTOX = 0.04

The toxicity indices calculated for each of the samples are presented in Figure 4.

This global index can thus give a quick indication to the operator of a station to intervene in the actual treatment of effluents.

Claims (13)

1. Method for estimating the toxicity of an aqueous sample comprising the following successive steps: distribution of different fractions of the aqueous sample of a medium to be tested in at least eight measurement cells, preferably at least ten measurement cells, each containing a single, non-pathogenic microbial strain, said microbial strains belonging to a panel different microbial strains with decreasing toxicological resistance profiles, each microbial strain being previously assigned a coefficient called robustness index or toxicological resistance index (Ind-Rob) vis-à-vis molecules or conditions frequently encountered in said medium to be tested; - placement of measurement cells in a thermostatically controlled enclosure; - monitoring of the inhibition of the microbial respiratory activity of each fraction of sample for a given duration, of at least 15 minutes, preferably at least one hour, the final result and / or the kinetic results relating to each fraction , providing information on the respiratory activity of microorganisms, - determination of an overall toxicity index (Ind-TOX) of the aqueous sample in the presence of each microbial strain, taking into account the robustness indices of each strain. 2. Method according to claim 1, characterized in that each measurement cell is prepared according to the following successive steps: - isolation and culture of the selected microbial strain, and implementation thereof in said cell, - conditioning in said cell with addition of a protective agent for the microbial strain against lyophilization, - then freeze-drying of the microbial strain and conservation. 3. Method according to claim 2, characterized in that the lyophilization phase is preceded by a freezing phase in a temperature range from -20 ° C to -80 ° C, the lyophilization taking place at a temperature below -40 ° C under a pressure below 0.10 mbar. 4. Method according to any one of the preceding claims, characterized in that the monitoring of the metabolic activity of the microorganisms is carried out by means of an oxygen, redox or pH sensor present in the measurement cell. 5. Method according to any one of claims 1 to 3, characterized in that a marker of the metabolic activity of bacteria, such as a fluorescent and / or colored marker of the respiratory activity of said microorganisms is added in each measurement cell, the monitoring of the respiratory activity of said microorganisms being carried out by spectrophotometry. 6. Method according to claim 5, characterized in that the marker for the metabolic activity of bacteria is resazurin. 7. Method according to any one of the preceding claims, characterized in that the various microbial strains selected, in particular for an assessment of toxicity of a sample of effluent from urban or industrial wastewater or from natural water, are part of a panel of bacterial strains chosen from: Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Enterobacter helveticus, Proteus mirabilis, Budvicia aquatica, Zoogloea resiniphila, Rhizobium radiobacter, Thauera linaloolentis, Bosea vertrisii, Comamonas testosteroni, Comamonas denitrificans, Arthrobacter aurescens, Staphylococcus sp, Acinetobacter towneri, Rhodococcus rhodochrous, Corynebacterium pseudodiphtheriticum , Variovorax paradoxus, Staphylococcus auricularis, Sphingobium yanoikuyae, Cupriavidus necator, Paracoccus denitrificans, Bacillus subtilis subtilis, Paracoccus pantotrophus, Bacillus funiculus, Bacillus mycoides, sorted in decreasing order of resistance to toxicity. 8. Method according to any one of the preceding claims, characterized in that the individual results of the metabolic activity measurements such as the kinetics of respiratory activity of each of the strains, relative to a control, are expressed in rate of inhibition (Tx_lnh) of said strain, and said individual results are grouped together giving a toxicological imprint of the sample. 9. Method according to any one of the preceding claims, characterized in that each microbial strain being initially assigned a coefficient called the robustness index or toxicological resistance index (ranging from 0 to 1), an overall toxicological index of the sample is calculated according to the following formula (I): _{,, rrnv} Zn = i (Jnd_Rob _n xTx_lnh _n ) ina_T0X = ---————————-- Zn = l ( ^Ind - ^Rob n) (|) with x: number of microbial strains tested lnd_TOX: Index of global toxicity lnd_Rob _n : Robustness index of the strain n Tx_lnh _n : Inhibition rate of strain n 10. Kit for carrying out the evaluation of the toxicity of an aqueous sample, said kit comprising: - at least eight measurement cells, preferably ten measurement cells, each containing a single microbial strain, non-pathogenic, and lyophilized in the presence of a protective agent, preferably a control cell for the sample and a control cell for each microbial strain, - a marker for the metabolic activity of said bacteria, such as a fluorescent and / or colored marker, and - detailed instructions for implementing the method according to one of the preceding claims. 11. Kit according to claim 10, characterized in that the measuring cells 5 are glass vials, closed, preferably in the form of individual tubes or arranged in microplates. 12. Use of the method according to any one of claims 1 to 9, for the estimation of the toxicity of an effluent of urban wastewater, of effluent from the food industry or from industry, rainwater, surface water, surface or groundwater, or marine water. 13. Use according to claim 12, for monitoring treatment or purification channels, or environmental monitoring.