Use of a cnidarian-dinoflagellate symbiotic organism as a marker for evaluating the ecotoxicity of a product, and method for evaluating the ecotoxicity of a product

Abstract

The present invention relates to a use of a cnidarian-dinoflagellate symbiotic organism as a marker for evaluating the ecotoxicity of a product. The present invention also relates to a method for evaluating the ecotoxicity of a product, comprising the following steps: 1) culturing at least one cnidarian-dinoflagellate symbiotic organism in a suitable medium, 2) adding at least one product to the medium, 3) measuring, in the medium and the cnidarian, the dissociation of the dinoflagellates of the symbiosis and / or the growth rate of cnidarian cuttings, in particular hard coral cuttings.

Description

Description Title of the invention: USE OF A CNIDARIUS-DINOFLAGELLUM SYMBIOTIC ORGANISM AS A MARKER FOR EVALUATING THE ECOTOXICITY OF A PRODUCT, AND METHOD FOR EVALUATING THE ECOTOXICITY OF A PRODUCT Technical field

[0001] The present invention relates to a use of a cnidarian-dinoflagellate symbiotic organism, as well as to a method for evaluating the ecotoxicity of a product.

[0002] The present invention finds applications in particular in the field of toxicology, in particular ecotoxicology.

[0003] In the description below, the references in brackets ([ ]) refer to the list of references presented at the end of the text. State of the art

[0004] In recent years, it has been demonstrated that the marine environment is particularly impacted by the toxicity of certain products used in cosmetic products, particularly sunscreens. Indeed, some products can cause coral bleaching or even death. However, these organisms are already seriously affected by climate change. In this context, the cosmetics industry is increasingly subject to drastic regulations aimed in particular at ensuring the safety of its products with respect to the marine environment and more specifically cnidarians, a zoological group that includes tropical corals.

[0005] Cnidarian-dinoflagellate associations such as those forming reef-building corals are mutualistic symbioses of fundamental importance in the marine environment. These partnerships form the trophic and structural foundation of coral reef ecosystems. This intracellular association is centered on nutrient exchange and is essential for both partners to thrive in resource-poor tropical seas. nutrients. Cnidarian hosts, such as corals and anemones, harbor photosynthetic dinoflagellate endosymbionts, of the genus Symbiodinium, within gastrodermal cells in phagosomally derived vacuoles called symbiosomes. Initial colonization most often occurs when the host's gastrodermal cells lining the gastric cavity phagocytose symbionts ingested through the mouth during feeding.

[0006] Although cnidarian-dinoflagellate symbioses are stable under unstressed conditions, various environmental stressors, including elevated temperatures caused by global warming, can destabilize the partnership and lead to the loss of symbionts from host tissues. This phenomenon, known as cnidarian or coral bleaching, results in a significant reduction in host fitness, slowed growth, or even death, and can lead to the destruction of reefs. More recently, it has been shown that certain components used in cosmetic products can have disastrous environmental repercussions, including the bleaching of tropical corals.The democratization of low-cost air travel in recent years has exacerbated this phenomenon, as more and more people flock to tropical beaches each year, bordering coral reefs already weakened by climate change. Cosmetic products, particularly sunscreens, come into contact with corals in high concentrations during swimming.

[0007] When the cnidarian has bleached, it finds itself deprived of the food source provided by dinoflagellates. This results in the death of the cnidarian within a few weeks if it has not been recolonized by symbionts. Even low concentrations over the long term can have deleterious effects, notably, the slowing down of the mass gain of the coral. Generally, a species of coral living on the upper parts of the reef (e.g.: Acropora spp, Pocilopora spp, Seriatorpora spp, etc.), under stable physiological conditions, will gain up to 1% of its mass per day. This growth rate can significantly decrease if the species in question is subjected to any stress such as chronic exposure to a sublethal toxic substance. During these acute or chronic, it is an entire ecosystem that is weakened with very significant socio-economic consequences. Indeed, these ecosystems, apart from the economic windfall from tourism that they represent, provide local populations with a source of food and coastal protection. It is estimated today that 1 billion people on earth depend directly on coral reefs as their main source of food. It is therefore essential to urgently implement a global policy for these ecosystems and to better regulate the negative impact that abundant tourism can have, which carries a whole series of chemical products with harmful ecological consequences.

[0008] Thus, bioassays have been developed with the aim of evaluating the ecotoxicity of a product.

[0009] Some bioassays are commercially available as "ready-to-use" kits based on miniaturization and standardization of conventional toxicity tests (https: / / www.microbiotests.com / toxkit / ). These kits contain, in particular, test organisms, such as daphnia, rotifers, microaglues, etc., which can, for example, be in the form of dormant eggs in the case of daphnia (sporocysts). This form of commercialization has the advantage of freeing the user from the constraints of breeding and culture. It also makes it easy to have a variety of bioassays that can be used to constitute a minimal battery of tests to characterize the ecotoxicity of a sample. However, these bioassays are not very representative of reef ecology.

[0010] Other tests have been developed to assess the ecotoxicology of cosmetic products on corals, such as "ReetTox" (SGS, France). This aims to assess the toxicological impact of a cosmetic ingredient or a cosmetic product on a coral population representative of tropical / Indo-Pacific swimming areas (known as the "coral triangle"). Thus, this test assesses the toxicity of products on 12 or 50 species / specimens of hard and soft corals in order to obtain a true representation of the coral population. Although these bioassays represent a real advance in reef ecotoxicology, they have the disadvantage of being extremely expensive, time-consuming and cumbersome to implement. In addition, the methods for physicochemical analysis of water or biological (coral survival) measures used are only indirect measures of the stress caused. It is therefore difficult to know whether any mortality observed in the quarantine tank is due to exposure to the chemical tested or to the fact that the cnidarian was moved to another tank during the experiment. These organisms are indeed very sensitive and any change of environment can lead to bleaching or even mortality. In addition, bleaching of a cnidarian exposed to a toxic product can spread to other neighboring organisms not exposed to these same chemical compounds, resulting in a difficult, if not impossible, interpretation of the results.

[0011] Finally, other tests have been developed on cell cultures. For example, the VIRIS test (INNOV&SEA) is a cytotoxicity test on cell cultures from the anemone Anemonia viridis. This test is cumbersome to implement (14 days). Furthermore, this anemone lives in the cold waters of the Mediterranean and the Atlantic, which makes it an organism that is not very representative of the animals that inhabit coral reefs.

[0012] There is therefore a real need for testing to assess the toxicity, and in particular the ecotoxicity, of a product, particularly on coral reefs. Description of the invention

[0013] The present invention aims precisely to meet these needs and drawbacks of the prior art.

[0014] Following extensive research, the Applicant has succeeded in developing a standardized method for assessing the health status of cnidarians by measuring bleaching and / or growth rate.

[0015] The invention results in a simple, quick to implement, and inexpensive method, making it possible to highlight the suffering of cnidarians subjected to their exposure to chemical products such as those present in cosmetic products.

[0016] Advantageously, this process can be performed by a laboratory technician, and requires only a low weekly maintenance time, which can be as little as approximately 1 hour per week.

[0017] In addition, the method of the invention has high sensitivity. Advantageously, it can highlight sublethal signs of animal suffering such as coral bleaching or a decrease in growth rate. This allows the same coral cutting to be used successively in multiple bioassays.

[0018] Furthermore, the invention is particularly representative of the impact of chemical pollution on a coral reef.

[0019] Advantageously, the invention can be used on a large scale to assist in decision-making in order to combine economic development, product development, particularly cosmetics, and preservation of the marine environment.

[0020] Finally, the invention can make it possible to assess the health status of corals and to know whether they are subject to acute and / or chronic stress.

[0021] Thus, a first object of the invention relates to a use of a cnidarian-dinoflagellate symbiotic organism as a marker for the evaluation of the ecotoxicity of a product.

[0022] A second subject of the invention relates to a method for evaluating the ecotoxicity of a product, comprising the following steps: 1) cultivation of at least one cnidarian-dinoflagellate symbiotic organism in a suitable medium, 2) addition, in the environment, of at least one product, 3) measurement, in the environment and the cnidarian, of the dissociation of dinoflagellates from the symbiosis and / or the growth rate of cnidarian cuttings, in particular of “hard” corals belonging to the order of scleractinia.

[0023] Within the scope of the invention, any cnidarian-dinoflagellate symbiotic organism known to those skilled in the art may be used.

[0024] In particular, the cnidarian can be a "hard" coral (with a calcareous skeleton), a "soft" coral (without a calcareous skeleton), or an anemone. It can be a whole organism, a fragment (for example a cutting) of a whole organism, and / or cells in culture. For example, the "soft" cnidarian can be at least one cnidarian chosen from Aiptasia spp, Aiptasia pallida, Zoanthus spp, Xenia spp, Clavilaria spp. The "hard" coral can be an Acropora spp, Pocilopora spp, Seriatorpora spp, Stylophora spp, Turbinaria spp, Montipora spp. The dinoflagellate can be of the genus Symbiodinium, also called zooxanthellae.

[0025] Preferably, the cnidarian used for the step of measuring, in the medium and the cnidarian, the dissociation of dinoflagellates from the symbiosis, can be Aiptasia spp, and in particular Aiptasia pallida. It has characteristics that make it an excellent model, including rapid growth, easy maintenance conditions and its ability to be bleached and reinfected. It is possible to maintain large cultures of A. pallida symbiotic and aposymbiotic and many molecular and cellular biology techniques in Aiptasia are (Detournay et al., Dev Comp Immunol, 2012 ([1]), including a database of 12,000 ESTs of A. pallida sequences. A. pallida can range from less than 2 mm to 4-5 cm in height. It reproduces asexually via pedal laceration, in which motile polyps leave behind small pieces of the pedal disc that develop into tiny new polyps less than 1 mm in size.Large colonies (i.e., hundreds to thousands of individuals) of genetically identical animals can therefore be generated and maintained in culture indefinitely. A. pallida can be "bleached" by subjecting the individuals to a test product if it exhibits ecotoxicity. Within days or weeks, the polyps are free of algae (aposymbiotic), and if kept in the dark, they will remain aposymbiotic indefinitely.

[0026] Preferably, the cnidarian used for the growth rate measurement step may be the "hard" coral Seriatopora hystrix. It has particularly interesting characteristics for measuring growth rate. It is a fast-growing species that can be easily propagated by simple fragmentation, preferably in cuttings greater than 1 cm in length. These small cuttings will quickly heal and can therefore be used in growth rate assessment tests when exposed to potentially toxic products.

[0027] The product may be any substance or mixture of substances for which the potential toxicity to marine reef fauna is to be verified. In particular, it may be at least one product chosen from a chemical product and a biological product, for example a cosmetic product, a petrochemical product, a detergent product, an agri-food product or a phytosanitary product, in general. In particular, it may be a product used in the composition of a sunscreen.

[0028] In the context of the invention, the cnidarian-dinoflagellate symbiotic organism is used as a marker in that the measurement of the dissociation of the dinoflagellates from the symbiosis indicates its suffering, and therefore ecotoxicity, due to the presence of the product to be tested in the environment.

[0029] Step 1) of culture can be carried out by any method known to those skilled in the art allowing the symbiotic organism to multiply without time limitation. The culture conditions, in particular temperature, oxygen level, salinity, food, or light, are controlled and can be adapted by those skilled in the art depending on the symbiotic organism used and its quantity in the medium. The culture container can be any container commonly used for a bioassay, for example an aquarium, a microplate, a pot, beaker, petri dish, this list not being limiting. Advantageously, the culture can be carried out in a standardized manner in a multi-well plate.

[0030] For the purposes of the present invention, the term "suitable environment" means any environment that allows the multiplication of the cnidarian, without limitation of duration. This may be, for example, sea water or artificial sea water.

[0031] Step 2) of adding at least one product to the medium can be carried out by adding the at least one product at one or different known concentrations. The culture time can be adapted by a person skilled in the art so that the possible effects of the product(s) on the symbiotic organism can be observed and / or measured.

[0032] Advantageously, the at least one product can be introduced into the medium at different concentrations to obtain at least one threshold value chosen from the NOEC (no observed effect concentration), the LOEC (minimum observed effect concentration) and the EC50 (effective concentration). If different product concentrations are tested, the medium containing the symbiotic organism is divided from step 1) into as many samples as necessary. A negative control (i.e. without product) can also be produced to validate and / or interpret the results and establish one or more reference values that characterize the ecotoxicity level of a product or mixture of products. Several replicates of each of the tested concentrations can be carried out to ensure the accuracy and reliability of the results. Although not essential, it is also possible to introduce a positive control that can provide information on the maximum biological effect observed. The results of the samples should then be expressed in relation to this maximum observed effect.

[0033] As indicated above, the reference values can be the following: - the no observed effect concentration (NOEC): this is the highest concentration of substance / sample tested for which no effect significantly different from the control was observed; - the lowest observed effect concentration (LOEC): it corresponds to the lowest concentration for which an effect significantly different from the control was observed; - the effective concentration EC50 (in English: EC50): it corresponds to the concentration of substance / sample which causes an effect different by 50% compared to the control. The EC50 is determined statistically on the basis of modeling.

[0034] Step 3) of measuring, in the medium and the cnidarian, the dissociation of the dinoflagellates from the symbiosis, can be carried out by any method known to those skilled in the art. Advantageously, the measurement of the dissociation of the dinoflagellates from the symbiosis can comprise the measurement of the concentration of dinoflagellates dissociated from the symbiosis in the medium, and / or the measurement of the disappearance, for example by digestion, of the dinoflagellate present within a tissue of the cnidarian, for example by microscopic analysis of the tissue of the cnidarian, or counting the tissue dinoflagellates after grinding and direct deduction of the rate of expulsion and / or digestion of the dinoflagellate. This can involve, for example, the measurement of the concentration of symbiotic dinoflagellate microalgae in the culture supernatant. This concentration can be determined, for example, using either a microscope and a counting chamber, or a plankton counter, or a spectrophotometer.

[0035] The measurement can be carried out at a frequency allowing the detection of acute stress, particularly measurable from one day to the next.

[0036] Advantageously, the assessment of acute stress can be carried out by measuring, in the medium and the cnidarian, the dissociation of the dinoflagellates from the symbiosis, optionally followed by a measurement of the expression of at least one gene known as a stress indicator, for example HSP (heat shock protein) and / or TGFbeta. Advantageously, the culture supernatant can then be removed and the cnidarians digested in order to determine the quantity of biomass by measuring total proteins or the quantity of microalgae resident in the animal. An advantage is that it is then possible to standardize the rate of microalgae expelled in the culture supernatant, in particular by relating this result to the animal biomass (for example by measuring total proteins) or to the number of total microalgae, corresponding to the quantity of dissociated microalgae plus the quantity of microalgae in the animal.It is also possible to deduce from these results the no observed effect concentration (NOEC), the lowest observed effect concentration (LOEC), and the effective concentration EC50.

[0037] To investigate acute or chronic stress, it is possible to measure the growth rate of cuttings, for example, cuttings of about 1 cm, of hard corals, by measuring the submerged mass, optionally followed by a measurement of the expression of at least one gene known as a stress indicator, for example HSP (heat shock protein) and / or TGFbeta. This submerged mass measurement can be carried out by attaching a submerged cutting to a thread, for example a fishing line, itself placed on the hook of an analytical balance. By taking into account the density of seawater, the density of the coral limestone skeleton, and the Archimedes' buoyancy force, the mass of the coral skeleton can be determined very precisely.

[0038] An increase in the dissociation of dinoflagellates from the symbiosis, compared to the value measured at the beginning of step 2) or compared to the control, or a growth rate lower than that of the control, implies the existence of ecotoxicity of the product.

[0039] Optionally, the method of the invention may further comprise an analysis of the expression of at least one genetic stress marker by the cnidarian, for example by QRT-PCR, and / or an analysis of the growth rate, in particular of hard corals, by measurement of submerged mass, in particular to detect chronic stress evolving over several weeks, or even months.

[0040] Advantageously, the method of the invention may comprise a step of comparing the value obtained in step 3) with a value predicted or measured in a natural environment, making it possible to evaluate the ecotoxicity linked to the product. In this case, it is also possible to compare the threshold values possibly obtained with the concentrations predicted or measured in the natural environment to evaluate the ecotoxicity linked to the product studied.

[0041] Possibly, when cuttings are used, they can be previously acclimatized in a nursery, under lighting and temperature conditions identical to those to which they are subjected during steps 1) to 3) of the process. Advantageously, the acclimatized cuttings exhibit a stable physiological growth rate during an acute or chronic test campaign, not varying by more than 20% between the beginning and the end of the experiment. Thus, such prior acclimatization makes it possible to obtain a robust result, representative of natural conditions.

[0042] Advantageously, acclimatized cuttings can exhibit a physiological growth rate (corresponding to the negative control) not varying by more than 20% between the start and the end of the experiment (whether acute or chronic stress).

[0043] Advantageously, the cuttings can be placed, after step 3), in a rinsing bath composed of sea water, then placed back in the nursery, then possibly reused after a minimum period of one month, the time necessary for them to recover a physiological growth rate.

[0044] Other advantages may still appear to those skilled in the art upon reading the examples below, illustrated by the attached figures, given for illustrative purposes only. illustrative. Brief description of the figures:

[0045] Figure 1 represents a curve fitting to the experimental results, of the percentage of bleaching as a function of the product tested (pg / L, log scale).

[0046] Figure 2 represents an aquaculture device (RAS: recirculating aquaculture system) necessary for the acclimatization of coral cuttings prior to their use in a bioassay, comprising a container for the culture of at least one cnidarian-dinoflagellate symbiotic organism in a suitable medium, equipped with a biological filter (live rocks), a water upwelling pump, a physical filter (skimmer), as well as nurseries in which the cuttings are suspended, and LED lighting. EXAMPLES OR METHODS OF IMPLEMENTATION

[0047] Example 1: implementation of the method of the invention with A. pallida

[0048] Cultivation of A. pallida

[0049] A. pallida stock cultures (symbiotic) are maintained in aquaria at room temperature (i.e., approximately 24°C) and 60 pmoles quanta / m2 / sec on a 12-hour light / dark cycle. Stock cultures are fed twice weekly with brine shrimp nauplii. Before the start of an experiment, cnidarians are removed from the aquaria and placed in individual wells of multiwell plates in artificial seawater (ASW). The plates are placed in an incubator at 24°C, 60 pmoles quanta / m2 / sec on a 12-hour light / dark cycle, and allowed to acclimate for 1 to 2 days before experimentation. The anemones are then subjected to several treatments.

[0050] Carrying out the tests

[0051] The tests are carried out in 6 replicates. Each column of 6 wells (6 replicates) is subjected to a specific concentration of the product to be tested.

[0052] The first column contains no substance and therefore serves as a negative control (T-), while the second column serves as a positive control (T+) in which the cnidarians are subjected to a precise concentration of a substance known to induce cnidarian bleaching, copper sulfate (CuSO4, 5H2O) at 50-100 pg / L). Then, from column 3 to 8, the cnidarians are subjected to decreasing concentrations of the test product.

[0053] The plate is placed in a climatic chamber for 18 hours (200 PAR lighting; temperature 28°C).

[0054] At the end of this incubation period, the concentration of symbiotic dinoflagellate microalgae in the culture supernatant is determined using either a microscope and a counting chamber, or the HydraL (a planktonic counter developed by PLANKTOVIE sas), or a spectrophotometer.

[0055] Then, the supernatant from the wells is removed. The cnidarians from each well are then digested to determine the amount of biomass by measuring total protein (e.g., Bradford Assay) or the amount of microalgae resident in the animal.

[0056] Thanks to this, it is now possible to standardize the rate of microalgae expelled into the culture supernatant per unit of animal protein, or per total quantity of microalgae per well (dissociated microalgae + microalgae in the animal). From these results, we deduce • the no observed effect concentration (NOEC), • the minimum observed effect concentration (MOEC), • the effective concentration EC50 (in English: EC50)

[0057] Comparing these threshold values with the concentrations predicted or measured in the natural environment makes it possible to assess the environmental risk linked to the product studied.

[0058] Additional biomolecular tests can be carried out on animals subjected to these threshold concentrations, such as measurements of the expression of certain genes known as indicators of stress, for example HSP (heat shock protein) and / or TGFbeta.

[0059] Example 2: implementation of the method of the invention with Seriatopora hystrix

[0060] Evaluation of the mass gain of hard corals, type Seriatopora hystrix (scleractinian).

[0061] Approximately 1 cm long cuttings of S. hystrix (symbiotic) are grown from stock colonies. These cuttings are then attached to a small fishing line (10 cm long) and suspended in the water column of aquaria at room temperature (approximately 24°C) and 60 pmoles quanta / m2 / sec on a 12-hour light / dark cycle. These cuttings must be fully healed before they can be used in further bioassays. This is usually the case after 1-2 weeks. Before starting an experiment, cnidarians are removed from the aquaria and placed in individual wells of multiwell plates in artificial seawater (ASW). The plates are placed in an incubator at 24°C, 60 pmoles quanta / m2 / sec on a 12-hour light / dark cycle and allowed to acclimate for 1-2 days before experimentation. The cuttings are then subjected to several treatments.

[0062] Carrying out the tests

[0063] Tests are performed in 3-6 replicates. Each column of 6 wells (6 replicates) is subjected to a specific concentration of the product to be tested.

[0064] The first column contains no substance and therefore serves as a negative control (T-), while the second column serves as a positive control (T+) in which the cnidarians are subjected to a specific concentration of a substance known to decrease coral growth, copper sulfate (CuSO4, 5H2O) at 5-10 pg / L. Then, from column 3 to 8, the cnidarians are subjected to decreasing concentrations of the test product.

[0065] The plate is placed in a climatic chamber for the duration of the chronic stress assessment, typically over a period of 1-2 months (200 PAR lighting; temperature 28°C). Each week, the culture medium in each well is replaced with fresh medium containing the substance to be tested.

[0066] Also every week, an immersed mass measurement is carried out using an analytical balance equipped with a suspension hook.

[0067] The fishing line to which the cutting is attached is placed on the hook of the analytical balance, and the mass of the cutting is recorded.

[0068] From these results we deduce • the no observed effect concentration (NOEC), • the minimum observed effect concentration (MOEC), • the effective concentration EC50 (in English: EC50)

[0069] Comparing these threshold values with the concentrations predicted or measured in the natural environment makes it possible to assess the environmental risk linked to the product studied.

[0070] Additional biomolecular tests can be carried out on animals subjected to these threshold concentrations, such as measurements of the expression of certain genes known as indicators of stress, for example HSP (heat shock protein) and / or TGFbeta.

[0071] Example 3: implementation of the method of the invention with a panel of hard corals representative of tropical reef fauna.

[0072] Cuttings of about 1-2 cm in length of scleractinia (Montipora spp, Acropora spp, Seriatora spp, Pocillopora spp, Stylophora spp) are taken from mother colonies. These cuttings are then attached to a fishing line (10 cm long) and suspended in a nursery for a minimum period of one month before being used in bioassays.

[0073] It is important that the nursery is placed in a closed water circuit (RAS: recirculating aquaculture system) whose stability of the complete system is ensured by a physical filter (skimmer), a biological filter composed of live rock, LED lighting (60 pmoles quanta / m2 / sec), heating (24-27°C), temperature control probes, pH, etc. (Fig.2). This complete system is composed of artificial seawater made from salts enriched with calcium and carbonate hardness (KH) (e.g. Aqua Forest Reef Plus). After one month, the level of healing of the cuttings must be complete before they can be used.

[0074] To carry out the bioassays, the cuttings are suspended in a transparent container (e.g., a 2L beaker), with 3 cuttings per beaker, filled with the water to be tested. For each beaker, the water to be tested is obtained by taking 2L of water from the complete system and solubilizing a substance X to be tested by stirring with a magnetic bar for 24 hours. Afterwards, each solution is filtered through a 0.7 pm filter using a vacuum pump. The solution obtained, brought to 25°C, can be used for beaker tests, by immersing the cuttings whose mass has been previously determined by submerged weighing. This solution is replaced every 3 days with a new solution prepared as described above.

[0075] It is important to place the beakers throughout the duration of the acute (24-72H) or chronic (2-4 weeks) test in light and temperature conditions similar to the nursery, typically 300 PAR lighting; temperature 25°C.

[0076] At the end of the acute test, or each week during the chronic test, an immersed mass measurement is carried out using an analytical balance equipped with a suspension hook.

[0077] The fishing line to which the cutting is attached is placed on the hook of the analytical balance, and the mass of the cutting is recorded.

[0078] From these results we deduce • the no observed effect concentration (NOEC), • the minimum observed effect concentration (MOEC), • the effective concentration EC50 (in English: EC50)

[0079] Comparing these threshold values with the concentrations predicted or measured in the natural environment makes it possible to assess the environmental risk linked to the product studied.

[0080] Additional biomolecular tests can be carried out on animals subjected to these threshold concentrations, such as measurements of the expression of certain genes known as indicators of stress, for example HSP (heat shock protein) and / or TGFbeta.

[0081] After the tests, the cuttings are placed in a rinsing bath composed of seawater prepared under the same conditions as those described above. Then, these same cuttings are placed back in the nursery. They can be reused for new tests after a minimum period of one month, the time necessary for them to recover a physiological growth rate. REFERENCES 1. Detournay et al.: “Regulation of cnidarian-dinoflagellate mutualisms: Evidence that activation of a host TGFp innate immune pathway promotes tolerance of the symbiont”, Dev Comp Immunol. 2012 Dec;38(4):525-37.

Claims

Claims

1. Use of a cnidarian-dinoflagellate symbiotic organism as a marker for the evaluation of the ecotoxicity of a product.

2. A method for evaluating the ecotoxicity of a product, comprising the following steps: 1) cultivation of at least one cnidarian-dinoflagellate symbiotic organism in a suitable medium, 2) addition, in the environment, of at least one product, 3) measurement, in the environment and the cnidarian, of the dissociation of dinoflagellates from the symbiosis and / or the growth rate of cnidarian cuttings, particularly hard corals.

3. Method according to claim 2, wherein said at least one product is introduced into the medium at different concentrations to obtain at least one threshold value chosen from the CSEO (concentration without observed effects), the CMEO (minimum concentration with observed effects) and the EC50 (effective concentration).

4. Method according to claim 2 or 3, comprising a step of comparing the value obtained in step 3) with a value predicted or measured in a natural environment, making it possible to evaluate the ecotoxicity linked to the product.

5. Method according to any one of claims 2 to 4, in which the culture is carried out in a standardized manner in a multi-well plate.

6. A method according to any one of claims 2 to 5, wherein measuring the dissociation of dinoflagellates from the symbiosis comprises measuring the concentration of dinoflagellates dissociated from the symbiosis in the medium and / or measuring the disappearance, for example by digestion, of the dinoflagellate present within a tissue of the cnidarian, for example by microscopic analysis of the tissue of the cnidarian, or counting the tissue dinoflagellates after grinding, and directly deducing the rate of expulsion and / or digestion of the dinoflagellate.

7. Method according to any one of claims 2 to 6, further comprising an analysis of the expression of at least one genetic stress marker by the cnidarian, for example by QRT-PCR, and / or an analysis of the growth rate of the cnidarian by measurement of submerged mass.

8. Use according to claim 1, or method according to any one of claims 2 to 7, characterized in that the cnidarian is at least one cnidarian chosen from Aiptasia spp, Zoanthus spp, Xenia spp, Clavilaria spp., Acropora spp, Pocilopora spp, Seriatorpora spp, Stylophora spp, Turbinaria spp, and Montipora spp.

9. Use or method according to claim 8, characterized in that the cnidarian is a whole organism, a fragment of a whole organism, or cells in culture.

10. Use or method according to claim 8 or 9, characterized in that said at least one product is chosen from a chemical product and a biological product, for example a cosmetic product, a petrochemical product, a detergent product, an agri-food product or a phytosanitary product.

11. Method according to claim 2, in which the cuttings are previously acclimatized in a nursery, under lighting and temperature conditions identical to those to which they are subjected during steps 1) to 3) of the method.

12. Method according to claim 11, wherein the acclimatized cuttings exhibit a physiological growth rate (negative control) not varying by more than 20% between the start and the end of the experiment (acute or chronic).

13. Method according to claim 11, wherein the cuttings are, after step 3), placed in a rinsing bath composed of sea water, then placed back in the nursery, then possibly reused after a minimum period of one month.

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