The present invention relates to a method for measuring the degree of toxicity of environmental samples due to the presence of bioavailable inorganic and organic chemical pollutants quickly, easily, at low cost and without using living organisms. It can be used particularly but not exclusively in the following sectors: waste management and treatment, water resource management and treatment, natural resource monitoring and management, sustainable agriculture, pollution control and depollution technologies, industrial processes with low environmental impact, production cycle quality certification, environmental decontamination.

As is known, human production activities can have a considerable impact on the surrounding environment. In particular, activities in the field of agriculture and industrial production can lead to the release of dangerous substances into the environment, such as for example heavy metals and xenobiotics (pesticides, polychlorinated biphenyls (PCB), aromatic polycyclic hydrocarbons and compounds such as diethyl phthalate, tetrabromobisphenol, hexabromocyclododecane, which are known to interfere with the endocrine system of animals and particularly of humans (endocrine disruptors). It is therefore important to monitor the quantity of these toxic substances in the environment, in order to avoid an accumulation thereof that is so high as to be able to damage flora, fauna and human beings.

For this purpose, environmental samples such as water, soil and sediments, as well as wastewater deriving from industrial production cycles and percolates, are subjected to tests to assess the degree of toxicity, on the basis of the results of which it is possible to formulate judgments regarding general toxicity, for example by following the guidelines issued by appointed authorities. The traditional toxicity tests commonly used are performed on living organisms. To assess the toxicity of fresh water, the most widely used test is the acute toxicity test using Daphnia magna; to assess the toxicity of sea water, various tests are available, such as the 24-hour mortality test with Brachionus plicatilis or the spermiotoxicity test with Paracentrotus lividus. To assess soil toxicity, the acute and chronic toxicity test with Eisenia fetida is used. The principle on which the execution of the above-cited tests is based consists in exposing, under standardized laboratory conditions, the test species to the environmental sample to be examined and in measuring specific parameters (such as mortality in the acute test with Daphnia magna, in the acute test with Brachionus plicatilis and in the acute test with Eisenia fetida, or inhibition of the capacity to fertilize eggs in the test with Paracentrotus lividus) after a certain exposure time. The execution times for tests based on the use of living organisms vary depending on the species. For example, in the acute toxicity test with Brachionus plicatilis the exposure time of the test species to the sample to be analyzed is 24 hours, following a time interval (generally 28 hours) needed for the dormant eggs to hatch.

In case of the spermiotoxicity test, the specimens of Paracentrotus lividus must be sampled by immersion in a control site, which therefore is not contaminated. Then the sampled specimens are stimulated to emit gametes. The spermatozoa are incubated for one hour with the aqueous medium to be analyzed and then finally placed in contact with the eggs for 20 minutes. The count of fertilized eggs with respect to non-fertilized ones by microscope observation allows measuring of the spermiotoxicity of the sample. In the case of the test with Eisenia fetida the time of exposure to the soil being studied is 15 days.

The use of living organisms entails disadvantages, such as long times and high costs of execution to procure the animals and keep them under laboratory conditions. Moreover, execution of the test is limited by the availability of the species under the physiological conditions suitable for execution of the test. For example, in the case of the test with Paracentrotus lividus, test execution is limited only to the reproductive periods of the species in the course of the year.

Moreover, another limitation of toxicological tests based on the use of living organisms is the possibility that the toxicity measurement might be biased by any presence, in the sample to be analyzed, of allelopathic substances and/or of organisms that are pathogenic for the species itself.

Furthermore, in tests based on the use of living organisms, particular chemical characteristics of the sample, such as pH and osmolarity, must be modified so that they are adequate for the physiological conditions of the test species.

This alteration of the chemical characteristics of the environmental sample can entail alterations in the bioavailability of the chemical pollutants that are present in the sample itself.

There is, therefore, the need to provide a method for assaying the degree of toxicity of an environmental sample that does not entail the use of living organisms. Moreover, since under most environmental chemical contamination conditions multiple chemical pollutants of various kinds are present simultaneously, such as heavy metals and xenobiotics, there is the need to provide a method that allows assessing both the general toxicity of an environmental sample and the amount of general toxicity due to some classes of chemical pollutants. Moreover, there is the need to provide a method that is at the same time capable of yielding results that can be referred directly to the possible effects of a toxic environmental sample on human health and can be performed quickly, easily and at low cost.

The aim of the present invention is to provide a method that allows assessing not only the general toxicity of an environmental sample but also the amount of general toxicity due to the presence of non-metallic chemical pollutants in the sample. Within the scope of this aim, an object of the invention is to provide a method for assessing the toxicity of of environmental samples that does not use living organisms for this assessment.

More particularly, an object of the invention is to provide a method for assessing the toxicity of environmental samples the results of which can be compared with those obtained with toxicity assessment methods based on the use of living organisms.

Another object of the invention is to provide a method for assessing the toxicity of environmental samples the results of which can be referred easily to potential effects on human health.

Another object of the present invention is to provide a method for the assessment of the toxicity of environmental samples that can be used for environmental monitoring in various fields, such as: waste management and treatment, water resource management and treatment, natural resource management, sustainable agriculture, pollution control and depollution technologies, industrial processes with low environmental impact, certification of the quality of production cycles, environmental decontamination.

Another object of the invention is to provide a method for the assessment of the toxicity of environmental samples that is highly reliable, relatively easy to provide and has competitive costs.

This aim, as well as these and other objects that will become more apparent hereinafter, are achieved by a method for assessing the toxicity of an environmental sample, comprising the steps of:

a) mixing an aqueous buffer solution with an aqueous solution of the enzyme carbonic anhydrase;

b) adding to the mix obtained in step (a) an aqueous solution comprising carbon dioxide (C0 ₂ ) or bicarbonate (HC0 ₃ -) so as to start the carbonic anhydrase enzyme reaction; c) determining the enzyme activity of the carbonic anhydrase by assessing over time the pH variation of the mix obtained in step (b), which is an indicator of the hydration reaction of CO ₂ or of the reverse dehydration reaction of HC0 ₃ ^_ , catalyzed by carbonic anhydrase;

d) repeating the procedure of steps (b) and (c) on the buffer solution of step (a) to which the aqueous solution of carbonic anhydrase has not been added, in order to measure non-specific activity;

e) repeating the procedure of steps (b) and (c) on the mix obtained in step (a), in which the solution comprising carbonic anhydrase has been preincubated beforehand with the environmental sample whose toxicity is to be assessed;

f) calculating the inhibition value (I) of carbonic anhydrase as a percentage ratio between (i) and (ii), where (i) is the difference between the enzyme activity of carbonic anhydrase determined in step

(e) and the non-specific activity determined in step (d) and (ii) is the difference between the enzyme activity of carbonic anhydrase determined in step (c) and the non-specific activity determined in step (d).

Further characteristics and advantages of the invention will become more apparent from the detailed description that follows and from the accompanying drawings, wherein:

- Figures 1A and IB are two charts of the variation of pH over time in the presence and in the absence of the carbonic anhydrase enzyme in the case of the hydration reaction of CO ₂ (Figure 1A) and of the dehydration reaction of HCO ₃ ' (Figure IB). In particular, the charts in the boxes show in detail the linear trend of pH variation during the first 30 seconds of reaction;

- Figures 2A and 2B are two charts of the dose-response curve, which indicate the percentage of inhibition of the activity of carbonic anhydrase as a function of the concentration of heavy metals (CdCl ₂ , CuCl ₂ , HgCl ₂ , Figure 2A) or of pesticides (carbaryl, malathion, Figure 2B) in an environmental sample;

- Figures 3A and 3B are two charts that plot the values of IC ₂₀ (Figure 3A) and of IC ₅₀ (Figure 3B) expressed in g/1 for environmental samples of fresh water and sea water containing various chemical pollutants. The pollutants are listed in order of decreasing toxicity for the fresh water sample.

The method described herein consists in a procedure that is useful for measuring the degree of toxicity of environmental samples due to organic and/or inorganic chemical pollutants and is based on the in vitro assessment of the degree of inhibition of catalytic activity of the carbonic anhydrase enzyme.

The degree of inhibition of the enzyme activity of carbonic anhydrase is directly proportional to the concentration of bioavailable toxic substances present in the analyzed environmental sample.

In the context of the present invention, the term "general toxicity" of an environmental sample is understood to refer to the capacity of said sample to cause harmful effects to living organisms in relation to the simultaneous presence therein of multiple bioavailable chemical pollutants (both organic and inorganic), which, together, can have an overall (additive and/or synergistic) toxic effect on biological systems.

In the context of the present invention, moreover, the term "environmental sample" is understood to refer to any environmental component of natural origin that can store chemical pollutants. For example, the environmental sample can be constituted by fresh water, sea water or salt water, interstitial water, wastewater, percolates, elutriates of sediment, elutriates of soil and extracts. The chemical substances capable of causing toxic effects can be both inorganic substances (for example heavy metals) and organic substances (for example pesticides, polychlorinated biphenyls (PCB), aromatic polycyclic hydrocarbons and compounds such as diethyl phthalate, tetrabromobisphenol, hexabromocyclododecane). These last compounds are currently receiving increasing attention, since it appears that they have an endocrine disruptor effect, i.e., they are capable of interfering with the endocrine system of animals and of humans in particular.

Moreover, in the context of the present invention, the term "nonspecific activity" is understood to refer to the number of micromoles of H ⁺ ions produced in one minute by the spontaneous hydration reaction (i.e., not catalyzed by carbonic anhydrase) of C0 ₂ or, vice versa, consumed in one minute by the spontaneous dehydration reaction (i.e., not catalyzed by carbonic anhydrase) of Non-specific activity generates a variation of pH associated with the spontaneous (non-catalyzed) hydration reaction of C0 ₂ or dehydration reaction of HC0 ₃ ^" .

Carbonic anhydrase is an enzyme that is present in animals, plants and bacteria, capable of catalyzing both the hydration reaction of carbon dioxide (C0 ₂ ) to carbonic acid (H ₂ C0 ₃ ), which in an aqueous environment is present in dissociated form, as bicarbonate ions (HC0 ₃ -) and protons, and the reverse reaction of dehydration of bicarbonate to carbon dioxide:

C0 ₂ + H ₂ 0 <→ H ₂ C0 ₃ <→ HC0 ₃ - + H ⁺

The substrates of carbonic anhydrase are therefore carbon dioxide (C0 ₂ ) and bicarbonate (HC0 ₃ ).

Carbonic anhydrase belongs to the class of metalloenzymes, since its catalytic site is characterized by the presence of a metal ion, generally a zinc ion, coordinated with the imidazole rings of three histidine residues.

In vivo, the role of carbonic anhydrase is linked mainly to control of the acidity of biological fluids, for example blood, by conversion of carbon dioxide to carbonic acid.

The determination of the enzyme activity of carbonic anhydrase, necessary to calculate the inhibition value of said enzyme, is performed by measuring the initial rate of the reaction catalyzed by carbonic anhydrase. This determination is performed according to a procedure that is simple, reliable and low-cost and consists in monitoring the pH variation of the mix prepared in step (b) of the method by means of a method of the electrometric or colorimetric type.

The pH variation is a decrease in case of the hydration reaction of C0 ₂ and an increase in case of the dehydration

It has been mentioned that monitoring of the pH variation in the mix can be performed both electrometrically, by use of a pH meter, and colorimetrically, by measuring the absorbance of the mix to which a pH variation colorimetric indicator has been added. For example, it is possible to use the neutral red indicator (3-amino-7-dimethylamino-2- methylphenazine hydrochloride, also known as toluylene red) at a concentration preferably comprised between 2 and 5 μΜ. It is noted that neutral red has never been used so far to measure the enzyme activity of carbonic anhydrase. The use of a colorimetric indicator of pH variation can be applied both in the hydration reaction of C0 ₂ and in the dehydration reaction of As the pH in the mix varies, so does the absorbance of said mix. By interpolation on a calibration line in which the variation of absorbance of the dye as a function of pH is expressed, it is possible to convert the colorimetric measurement of absorbance into a pH measurement. The presence of neutral red, at the indicated concentrations, in the mix whose pH variation is assessed has no non-specific effect on the activity of carbonic anhydrase.

It has been mentioned that in order to determine the enzyme activity of carbonic anhydrase the initial rate of the reaction catalyzed thereby is measured. The initial rate, which corresponds to the rate of the enzyme reaction in its initial steps, can be calculated from the slope of the line that interpolates the experimental data related to pH variation over time, preferably within the first 15-30 seconds from the beginning of the reaction (see Figures 1A and IB), i.e., when the pH variation over time shows a linear behavior. The slope of the line, indicated by (a) and expressed as ΔρΗ/min, can be calculated both for the hydration reaction of C0 ₂ and for the dehydration

Therefore, the measurements of the electrometric and colorimetric type described here can be used to assess the activity of carbonic anhydrase in terms of both hydration of C0 ₂ and dehydration of bicarbonate. In the first case, one measures the increase in hydrogen ions (H ⁺ ), i.e., the pH decrease, in the mix prepared in step (b) of the method. The increase in H ⁺ ions is due to the forming of carbonic acid in the reaction catalyzed by the enzyme over time. In the second case, one measures the decrease in hydrogen ions (H ⁺ ), i.e., the increase in pH, in the mix prepared in step (b) of the method. The decrease in H ⁺ ions is due to the forming of C0 ₂ and water in the dehydration reaction of bicarbonate catalyzed by the enzyme.

Enzyme activity, expressed as micromoles of product developed per minute, is calculated by multiplying the above-cited value (a) by the buffer capacity of the reaction medium (indicated by (β)), where "buffer capacity" is the quantity of acid or base (expressed in mmol/1) that must be added to said solution in order to alter its pH by one unit, by the volume (V) of the mix, according to the formula:

A = α * β * V

where the catalytic activity A is expressed in enzyme units.

The method according to the invention entails, in step (a), preparing a mix comprising an aqueous buffer solution and a solution of carbonic anhydrase. Preferably, the buffer solution has an initial pH comprised between 8.5 and 8.6 (or in any case higher than 8.0) if the hydration reaction of C0 ₂ is measured. If instead the dehydration reaction of bicarbonate is measured, the buffer solution has a pH preferably comprised between 6.8 and 7.0.

The buffer solution used in step (a) can comprise hydroxymethyl- aminomethane (2-amino-2-hydroxymethyl-propane-l ,3-diol or Tris) or 4-2- hydroxyethyl-l-piperazinyl-ethanesulfonic acid (HEPES) at concentrations comprised between 3 and 10 mM.

In some cases, in addition to the assessment of the general toxicity of an environmental sample, it can be useful to give an indication of the amount of toxicity due to non-metallic chemical pollutants. It is stressed that no currently available toxicity test is capable of indicating, within the same measurement, the percentage of toxic effect due to non-metallic contaminants with respect to the total toxic effect.

Therefore, in one embodiment of the present invention, the method can be performed by performing steps (a)-(e) as described above, wherein:

• a heavy metal chelating agent is also added to the mix of aqueous buffer solution and aqueous solution of carbonic anhydrase prepared in step (a); and

• step (e) is followed by step (f), in which the inhibition value of the carbonic anhydrase in the presence of the chelating agent (I _chel ) is calculated as a percentage ratio between (iii) and (iv), where (iii) is the difference between the enzyme activity of carbonic anhydrase determined in step (e) performed in the presence of the chelating agent and the non-specific activity determined in step (d) performed in the presence of the chelating agent and (iv) is the difference between the enzyme activity of carbonic anhydrase determined in step (c) performed in the presence of the chelating agent and the non-specific activity determined in step (d) performed in the presence of the chelating agent.

The inhibition value of the carbonic anhydrase in the presence of the chelating agent, therefore due to the presence of non-metallic compounds, is designated I _chel .

As is known in the background art, chelating agents are chemical compounds capable of forming complexes with certain metallic atoms or ions. Within the scope of the method according to the present invention it is possible to use the heavy metal chelating agents known commonly to the person skilled in the art, such as for example EGTA (ethylene glycol tetraacetic acid), EDTA (ethylene diamine tetraacetic acid), DTPA (diethylene triamine pentaacetic acid), BAPTA (l,2-bis(o- aminophenoxy)ethane acid).

Preferably, the heavy metal chelating agent is selected from the group constituted by EGTA and EDTA. Moreover, the concentration of the chelating agent is preferably comprised within 15 mM. At this concentration, the chelating agent does not interfere significantly with the activity of the enzyme and at the same time it is present in excess with respect to the metals contained in the environmental samples. This is very important, especially in the case of samples with a high calcium carbonate content (such as sea water or carbonate-rich fresh water), in which the calcium ions that are present in the sample bind the chelating agent that is present. The presence of chelating agent in excess ensures that it is possible to chelate the metallic cations that are present in the sample to be analyzed.

It is important to note that if general toxicity and its amount due to non-metallic chemical pollutants are assessed, the same enzyme isoform of carbonic anhydrase must be used both in the test performed in the presence of the chelating agent and in the test performed in the absence of the chelating agent. Likewise, in step (e) of the method, which provides for preincubation of carbonic anhydrase with the environmental sample, the preincubation time must be the same when the test is performed in the presence of the chelating agent and when the test is performed in the absence of the chelating agent.

Both for the test performed in the presence of chelating agent and for the test performed in the absence of chelating agent, if one uses C0 ₂ as a substrate of carbonic anhydrase its concentration in the mix that is the result of the addition of the aqueous solution mentioned in step (b) to the mix prepared in step (a) is comprised preferably between 100 and 200 mg/1. If instead one uses bicarbonate as a substrate of carbonic anhydrase, its concentration in the mix that is the result of the addition of the aqueous solution mentioned in step (b) to the mix prepared in step (a) is comprised preferably between 5 and 15 mM.

As mentioned earlier, it should be noted that the pH variation cannot be ascribed exclusively to the reaction catalyzed by carbonic anhydrase. A minimal part of H ⁺ ions is in fact produced also by the spontaneous hydration reaction of C0 ₂ ; likewise, a minimal part of H ⁺ ions is removed from the solution by the spontaneous dehydration reaction of the bicarbonate. However, since the rate of the spontaneous hydration reaction of C0 ₂ and of the spontaneous dehydration reaction of bicarbonate increases as the reaction temperature rises, by operating at temperatures comprised between 0°C and 10 °C it is possible to slow down the spontaneous reactions and minimize the contribution to pH variation due to the reactions not catalyzed by the enzyme. In this manner one obtains a higher sensitivity of the analytical method. In one embodiment of the present invention, therefore, steps (b), (c), (d) and (e) of the method can be performed at a temperature comprised between 0°C and 10°C.

As mentioned above, determination of the enzyme activity of carbonic anhydrase, which is performed in steps (c) and (e) of the method according to the invention, consists in measuring the initial rate of the reaction catalyzed by carbonic anhydrase, which corresponds to the slope of the line that interpolates the experimental data related to the pH variation over time, preferably in the first 30 seconds of reaction.

The method for measuring the activity of carbonic anhydrase according to the present invention is entirely innovative with respect to the methods known in the background art because:

1 ) it allows applying the same procedural scheme and the same calculation formula both to the C0 ₂ hydration reaction and to the bicarbonate dehydration reaction depending on the initial pH of the environmental sample, which can vary over a wide interval of values depending on the nature of said sample: sea water, fresh water, sediments, soils, wastewater, etc. In fact, depending on the initial pH of the sample, it is possible to choose the most appropriate mode (hydration reaction of C0 ₂ or dehydration of bicarbonate), which allows measurement of the toxicity of the sample under conditions that preserve the initial pH of the sample. For example, for samples with a pH of 8.0 or more, the choice of the hydration reaction of C0 ₂ is more appropriate; for samples with a pH of 7.5 or less, instead, it is preferable to use the dehydration reaction of bicarbonate. Therefore, with the method according to the invention it is not necessary to experimentally subject the environmental sample to great changes of its pH in order to adapt it to the conditions of execution of the test. This makes the test more reliable, since the bioavailability of the chemical contaminants in environmental matrices is influenced by various physical-chemical factors, one of the main ones being pH;

2) being based on the measurement of the initial rate of the enzyme reaction, the method according to the invention does not require a continuous supply of C0 ₂ during the enzyme reaction of hydration of

C0 ₂ , as instead required by most methods mentioned in the literature, which refer to the method described initially by Wilbur K.M. and Anderson G.N., "Electrometric and colorimetric determination of carbonic anhydrase", J. Biol. Chem., 176: 147-154, (1948);

3) it has a considerable versatility, since it lends itself to both electrometric and colorimetric measurements depending on instrument availability (pH meter or colorimeter) to the person who performs the test. This versatility makes the test executable at low cost even at non-specialized laboratories.

The method according to the present invention is effective with all commercially available forms of carbonic anhydrase. In a preferred embodiment of the method, the carbonic anhydrase used is a human isoform. Use of a human isoform of carbonic anhydrase allows obtaining information on the toxicity of environmental samples that can be related directly to potential effects on human health.

The time required for preincubation of the enzyme with the environmental sample whose toxicity is to be assessed varies depending on the type of sample to be examined but is generally less than 30 minutes.

The assessment of the toxicity of an environmental sample can furthermore require, in addition to the indication of the percentage of inhibition of enzyme activity, both general and due to non-metallic components, the assessment of the values of IC ₅₀ and of IC ₂₀ . These values are the values of concentration of toxic substances contained in the environmental sample that are capable of causing 50% and 20% enzyme inhibition respectively. In particular, the value of IC50 can be used to formulate a judgment on the toxicity of an environmental sample. The assessment of the value of IC ₂₀ is instead particularly important in toxicity tests, since it indicates the limits of detectability of toxicity for various chemical substances. The method according to the present invention offers therefore the possibility of determining in a simple and straightforward manner the values of IC ₅₀ and of IC ₂₀ for the environmental sample being examined.

In another aspect, the present invention relates to a kit for performing the method described herein. Said kit comprises the following components:

• an aqueous buffer solution;

• an aqueous solution comprising carbonic anhydrase;

• an aqueous solution comprising carbon dioxide (C0 ₂ ) or bicarbonate (HC0 ₃ ); and

• a device for the measurement of pH.

The device for the measurement of pH can be preferably a pH meter or a colorimetric indicator of pH variation. Preferably, the kit can comprise furthermore an aqueous solution comprising a metal chelating agent, if the kit is used to assess the amount of general toxicity of a sample due to non-metallic chemical pollutants.

It should be understood that the characteristics of the embodiments described with reference to the method according to the present invention are to be considered valid also in relation to the kit described herein even if they are not repeated explicitly.

The invention is now described further by means of examples, the content of which is to be considered non-limiting for the scope of the present invention.

EXAMPLE 1 : Measurement of the toxicity of real environmental samples by inhibition of carbonic anhydrase activity, determined both by means of the hydration reaction of C0 ₂ and by means of the dehydration reaction of bicarbonate.

In order to compare the reproducibility of the method, the assessment of enzyme activity was performed by utilizing both reactions catalyzed by carbonic anhydrase, i.e., hydration of C0 ₂ and dehydration of bicarbonate, on the same real environmental samples, represented by elutriate of three different marine sediments with a pH comprised between 7.5 and 8.0. The percentage of inhibition of carbonic anhydrase activity was measured by means of the procedure described above.

Table 1 lists the results obtained. The analysis was repeated three times for each sample. As can be seen from the table data, the two procedures return, for the same sample, results that closely overlap.

% inhibition of carbonic % inhibition of carbonic anhydrase anhydrase

(hydration of C0 ₂ ) (dehydration of HC0 ₃ )

Sample 1 1.5 ± 0.1 3.5 ± 0.3

Sample 2 62.1 ± 3.5 66.7 ± 3.6 Sample 3 | _. _ 73.4 ± 3.9 [ 73.8 ± 3.7 j

Table 1

EXAMPLE 2: Assessment of the inhibition of carbonic anhydrase by various chemical pollutants.

The method according to the invention was applied to the analysis of the toxicity of water samples containing increasing concentrations of heavy metals (in the form of CdCl ₂ , CuCl ₂ , HgCl ₂ and Na ₂ HAs0 ₄ ) and of a series of organic chemical pollutants that are representative of main classes of xenobiotics that are important for the assessment of environmental chemical contamination. In particular, the carbamate pesticide carbaryl, the organophosphate pesticide malathion, the polychlorinated biphenyl Aroclor, the aromatic polycyclic hydrocarbon phenanthrene and the endocrine disruptors diethyl phthalate (DEP), tetrabromobisphenol (TBBPA) and hexabromocyclododecane (HBCCD) were assessed.

Analysis of enzyme inhibition by the chemical pollutants listed above and measurement of the associated inhibition dose-response curves were performed both on fresh water samples and on sea water samples containing the compound whose toxicity was to be assessed.

By way of example, Figure 2A shows the inhibition dose-response curve of carbonic anhydrase for water samples containing CdCl ₂ , CuCl ₂ and HgCl ₂ . Figure 2B shows the inhibition dose-response curve of carbonic anhydrase for water samples containing carbaryl and malathion.

The inhibition percentage was determined as described previously in the text by using the hydration reaction of C0 ₂ . The inhibition values thus obtained are directly proportional to the toxic potential of the examined sample and a higher inhibition corresponds to a higher toxicity.

Moreover, the values of IC ₅₀ and of IC ₂₀ of the water samples containing the toxic compounds cited above were assessed by applying the method of the present invention. Figure 3 A shows the values of IC ₂₀ expressed in g/1 for the chemical pollutants listed earlier and assessed in two water samples: fresh water and sea water. The chart plots the compounds in order of decreasing toxicity (from the most toxic one, having the lowest IC ₂₀ , to the least toxic one, having the highest IC ₂₀ ), referring to the fresh water sample.

Figure 3B plots the values of IC ₅₀ expressed in g/1 for the chemical pollutants listed earlier and assessed in two water samples: fresh water and sea water. The chart plots the compounds in order of decreasing toxicity (from the most toxic one, having the lowest IC ₂₀ , to the least toxic one, having the highest IC ₂₀ ), referring to the fresh water sample.

As can be seen from the chart, the values of IC ₅₀ calculated for the various analyzed chemical pollutants were found to be different: this suggests, therefore, that the enzyme system used has a different degree of sensitivity with respect to the various pollutants analyzed. In particular, it is noted that the chemical pollutants that have a higher inhibition of the activity of carbonic anhydrase are malathion (organophosphate insecticide), Arochlor (PCB), carbaryl (carbamate insecticide) and phenanthrene. An intermediate effect is applied by heavy metals and by diethyl phthalate. A lower toxicity is associated with tetrabromobisphenol (TBBPA) and hexabromocyclododecane (HBCCD) (compounds used as flame retardants and having an endocrine disruptor effect) and with diphenyl.

The chart also demonstrates that carbonic anhydrase is sensitive to chemical compounds belonging to mutually heterogeneous classes.

As regards the values of IC ₂₀ , the high general sensitivity of the test to chemical compounds belonging to different classes is noted. In particular, for Arochlor it is noted that the value of IC ₂₀ is in the order of picograms/liter (1 * 10 ^-12 g/1) in fresh water.

In general, analysis of the inhibition of the enzyme at different salinity values shows that the sensitivity of carbonic anhydrase with respect to pollutants is on average higher in fresh water than in sea water. The explanation of this effect lies in that the characteristics of pH, hardness and ionic force of sea water can alter the concentrations of the various forms of the pollutant in solution and consequently modify its toxicity.

EXAMPLE 3: Assessment of the reliability of the method by correlation with standard tests for assessment of the general toxicity of real environmental samples.

In order to assess the reliability of the method according to the invention, a correlation analysis of the test based on inhibition of carbonic anhydrase activity with a test commonly used for toxicity assessment was performed.

This study allows an assessment of the reliability of the method according to the invention in the analysis of real environmental samples. The general toxicity of real environmental samples was thus assessed by means of the method according to the present invention and by means of standard tests commonly used in the background art and based on the use of living organisms. Thirty-seven environmental samples of different types, in particular elutriates of marine sediment and elutriates of soil, characterized both by a different chemical contamination and by a different salinity, were examined. The data obtained with the method according to the invention were correlated with the data obtained from the following tests:

• 24-hour mortality test with Brachionus plicatilis (ASTM Standard Guide El 440-91) on marine sediment elutriates;

• spermiotoxicity test with Paracentrotus lividus (Dinnel and Stober, 1987, Mar. Environ. Res., 21 : 121- 133) on marine sediment elutriates; · chronic test with Eisenia fetida worm on soil elutriates (OECD, 2004, Guideline for testing chemicals, No. 22, OECD, Paris, France).

The three tests were performed by working according to the methods and the conditions known to the person skilled in the art.

The results of the correlation analysis are given in the following Table 2, where: r Pearson is the Pearson correlation coefficient, P is the value of probability that the null hypothesis is true (absence of correlation) and n is the number of samples examined in each test:

24-hour mortality Spermiotoxicity Chronic test with test with test with Eisenia fetida Brachionus Paracentrotus

plicatilis lividus

Test with r Pearson=0.6807 r Pearson=0.6445 ! r Pearson=0.5923 carbonic P=0.0037 P=0.007 P=0.0424 anhydrase n=16 n=16 n=21

Elutriates of marine Elutriates of Elutriates of soil

I sediment marine sediment j

Table 2

As can be recognized from the table, in all three cases, for samples of different types, the correlation was found to be significant (P<0.05), a fact which confirms the reliability of the results obtained with the method according to the invention.

In practice it has been found that the in vitro analytical method according to the invention achieves fully the intended aim, since, if applied to an environmental sample affected by chemical contamination deriving from the simultaneous presence of multiple chemical pollutants, it allows measuring the general toxicity of the sample, i.e., the toxicity due to the simultaneous presence of multiple bioavailable chemical pollutants capable of having additive and/or synergistic effects on biological systems. Moreover, the method of the present invention allows measuring, in addition to the general toxicity of a sample, the amount of general toxicity due to the presence of non-metallic chemical pollutants, thus making it possible to determine the contribution of xenobiotics to general toxicity. In the case of chemical contamination deriving from the presence of a single pollutant, the measurement of the degree of inhibition of the enzyme makes it possible to deduce, by interpolation on appropriate dose-response curves, the concentration of bioavailable contaminant that is present in the sample.

Moreover, the method according to the invention achieves fully the intended aim, since it allows assessing the toxicity of environmental samples without using living organisms. It should be noted that the proposed method, being based on an in vitro enzyme measurement, offers the possibility to overcome one of the limitations of toxicological tests based on the use of living organisms, i.e., the possibility that the toxicity measurement might be biased due to the presence of allelopathic substances and/or of organisms that are pathogenic for the species on which the test is performed, can be present in the environmental sample to be examined and can influence the vitality of these species.

Moreover, from the correlation analysis between the results of the method according to the present invention and the analytical methods known in the background art listed in Table 2 it is possible to conclude that the enzyme method of the present invention is a quick and reliable system for assessing the degree of toxicity of environmental samples, the results of which are comparable with those of the tests currently in use, which are based on the use of living organisms.

It has also been found that the method of the present invention allows assessing the toxicity of environmental samples in short time and with reduced costs, thanks to the electrometric or colorimetric assessment of enzyme activity, which can be performed with a simple device for measuring the pH or with a colorimeter.

The method thus conceived is susceptible of numerous modifications and variations, all of which are within the scope of the accompanying claims; all the details may furthermore be replaced with other technically equivalent elements.

In practice, the materials used, as well as the dimensions, may be any according to the requirements and the state of the art. Where technical features mentioned in any claim are followed by reference signs, those reference signs have been included for the sole purpose of increasing the intelligibility of the claims and accordingly such reference signs do not have any limiting effect on the interpretation of each element identified by way of example by such reference signs.